Vaccines for blocking transmission of plasmodium vivax

ABSTRACT

The present invention relates novel methods and compositions for blocking transmission of  Plasmodium vivax  which cause malaria. In particular, Pvs25 and Pvs28 polypeptides, variants, including deglycosylated forms, and fusion proteins thereof, are disclosed which, when administered to a susceptible organism, induce an immune response against a 25 kD and 28 kD protein, respectively, on the surface of  Plasmodium vivax  zygotes and ookinetes. This immune response in the susceptible organism can block transmission of malaria.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a Continuation of U.S. Ser. No. 09/554,960,filed Feb. 12, 2003, which is a National Stage Entry of PCT/US98/25742,filed Dec. 4, 1998, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/067,596, filed Dec. 5, 1997; and U.S.Provisional Application Ser. No. 60/045,283, filed May 1, 1997. Theaforementioned applications are explicitly incorporated herein byreference in their entirety and for all purposes.

BACKGROUND OF THE INVENTION

Malaria continues to exact a heavy toll from mankind. Between 200million to 400 million people are infected by Plasmodium falciparum andPlasmodium vivax, the deadliest of the malarial protozoans, each year.One to four million of these people die.

Plasmodium vivax is probably the most prevalent form of malaria inhumans. It is rare in most of Africa because many Africans are Duffyblood group negative and P. vivax requires the Duffy blood group forinvasion. Although P. vivax does not often lead to death, it causesincomprehensible suffering and debilitation in hundreds of millions ofhumans every year.

The life cycle of the malaria parasite is complex. Infection in manbegins when young malarial parasites or “sporozoites” are injected intothe bloodstream of a human by a mosquito. After injection the parasitelocalizes in liver cells. Approximately one week after injection, theparasites or “merozoites” are released into the bloodstream to begin the“erythrocytic” phase. Each parasite enters a red blood cell in order togrow and develop. When the merozoite matures in the red blood cell, itis known as a trophozoite and, when fully developed, as a schizont. Aschizont is the stage when nuclear division occurs to form individualmerozoites which are released to invade other red cells. After severalschizogonic cycles, some parasites, instead of becoming schizontsthrough asexual reproduction, develop into large uninucleate parasites.These parasites undergo sexual development.

Sexual development of the malaria parasites involves the female or“macrogametocyte” and the male parasite or “microgametocyte.” Thesegametocytes do not undergo any further development in man. Uponingestion of the gametocytes into the mosquito, the complicated sexualcycle begins in the midgut of the mosquito. The red blood cellsdisintegrate in the midgut of the mosquito after 10 to 20 minutes. Themicrogametocyte continues to develop through exflagellation and releases8 highly flagellated microgametes. Fertilization occurs with the fusionof the microgamete and a macrogamete. The fertilized parasite, which isknown as a zygote, then develops into an “ookinete.” The ookinetepenetrates the midgut wall of the mosquito and develops into an oocyst,within which many small sporozoites form. When the oocyst ruptures, thesporozoites migrate to the salivary gland of the mosquito via thehemolymph. Once in the saliva of the mosquito, the parasite can beinjected into a host, repeating the life cycle.

Malaria vaccines are needed against different stages in the parasite'slife cycle, including the sporozoite, asexual erythrocyte, and sexualstages. Each vaccine against a particular life cycle stage increases theopportunity to control malaria in the many diverse settings in which thedisease occurs. For example, sporozoite vaccines would fight infectionimmediately after injection of the parasite into the host by themosquito. First generation vaccines of this type have been tested inhumans. Asexual erythrocytic stage vaccines would be useful in reducingthe severity of the disease. Multiple candidate antigens for this stagehave been cloned and tested in animals and in humans.

However, as drug-resistant parasite strains render chemoprophylaxisincreasingly ineffective, a great need exists for atransmission-blocking vaccine. Such a vaccine would block the portion ofthe parasite's life cycle that takes place in the mosquito or otherarthropod vector, thus preventing even the initial infection of humans.Several surface antigens serially appear on the parasite as it developsfrom gametocyte to gamete to zygote to ookinete within the arthropodmidgut (Rener et al., J. Exp. Med. 158: 976-981, 1983; Vermeulen et al.,J. Exp. Med. 162: 1460-1476, 1985). Several of these antigens inducetransmission-blocking antibodies.

The present invention fills the need for a means to completely blocktransmission of malaria parasites. The vaccine of the invention meetsthe requirements for a vaccine for controlling endemic malaria indeveloping countries: it induces high, long-lasting antibody titers, andcan be produced in large amounts, at the lowest possible cost.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to methods for preventing transmission ofmalaria, particularly Plasmodium vivax. The invention relates to methodsfor eliciting an immune response against parasites responsible formalaria. These methods comprise administering to a susceptible organisma pharmaceutical composition comprising Pvs28 polypeptides (such as SEQID NO:2), including partially or completely deglycosylated Pvs28polypeptides, Pvs25 polypeptides(such as SEQ ID NO:4), variants thereof,or Pvs25/Pvs28 fusion proteins(such as SEQ ID NO:5), in an amountsufficient induce an immune response against a 25 kD and 28 kD protein,respectively, on the surface of Plasmodium vivax zygotes and ookinetes.The immune response in the susceptible organism can block transmissionof malaria.

The invention also relates to methods of preventing transmission ofmalaria comprising administering to a susceptible organism apharmaceutical composition comprising a recombinant virus or expressioncassette encoding a Plasmodium vivax polypeptide, including Pvs28polypeptides (including partially or completely deglycosylated Pvs28polypeptides), Pvs25 polypeptides, or Pvs25/Pvs28 fusion proteins, in anamount sufficient to block transmission of the disease.

The invention further relates to pharmaceutical compositions comprisinga pharmaceutically acceptable carrier and a Pvs28 polypeptide(includingpartially or completely deglycosylated Pvs28 polypeptides), a Pvs25polypeptide, or a Pvs25/Pvs28 fusion protein, as described herein.

The invention also relates to isolated nucleic acids comprisingnucleotide sequences encoding Pvs28 polypeptides (including partially orcompletely deglycosylated Pvs28 polypeptides), Pvs25 polypeptides, orPvs25/Pvs28 fusion proteins. These nucleic acids may be isolated from,for instance, P. vivax. The sequences are typically contained in anexpression vector for recombinant expression of the proteins. Thesequences can also be incorporated into recombinant viruses, vectors orexpression cassettes for use as nucleic acid vaccines, including “nakedDNA” vaccines, for recombinant expression of the proteins in vivo. Inanother embodiment, the nucleic acids of the invention comprise apharmaceutical excipient and are injected into a host, e.g., as “naked”DNA vectors or “expression cassettes” injected into muscle, to expressrecombinant protein in vivo, to induce transmission blocking antibodiesagainst encoded polypeptides.

The invention also provides a composition comprising an isolated nucleicacid molecule encoding a Plasmodium vivax Pvs28 polypeptide lacking atleast one N-linked glycosylation site. In alternative embodiments, thenucleic acid encodes a polypeptide comprising a sequence as set forth inSEQ ID NO:2, excepting that the amino acid residue corresponding toresidue 130 of SEQ ID NO:2 is not an asparagine residue; and the aminoacid residue corresponding to residue 130 of SEQ ID NO:2 is glutamine.

The invention also provides a composition comprising an isolatedPlasmodium vivax Pvs28 polypeptide lacking at least one N-linkedglycosylation site. In alternative embodiments, the polypeptidecomprises a sequence as set forth in SEQ ID NO:2, excepting that theamino acid residue corresponding to residue 130 of SEQ ID NO:2 is not anasparagine residue; and, the amino acid residue corresponding to residue130 of SEQ ID NO:2 is glutamine.

The invention further provides a method of inducing a transmissionblocking immune response in a mammal, comprising administering apartially or completely deglycosylated Pvs28 polypeptide, or a nucleicacid encoding such a polypeptide, to a mammal.

Pvs28 (including partially or completely deglycosylated Pvs28) as animmunogenic carrier is provided for by the invention. Pvs28,administered with a second composition, provides a superior antigenicresponse to the second composition. Thus, the invention relates to animmunogenic composition capable of eliciting an immunogenic responsedirected to an epitope comprising an isolated Pvs28 and an isolatedmolecule comprising the epitope. The invention is also directed tomethods of eliciting an immunogenic response directed to an epitopecomprising administering an isolated Pvs28 and an isolated moleculecomprising the epitope. The Pvs28 and the second molecule can bechemically linked or joined together as recombinant fusion proteins.

In one embodiment, the Pvs28-containing fusion protein is a Pvs25-Pvs28fusion protein. The Pvs28 polypeptide can be designed to be partially orcompletely deglycosylated, as described herein. The sexual stagemalarial proteins Pvs25 and Pvs28, in the form of a Pvs25-Pvs28 fusionprotein, will generate transmission-blocking antibodies against bothPvs25 and Pvs28. These fusion proteins have enhanced antigenicproperties, as compared to use of either alone as an immunogen.

The invention also provides for Pvs25/Pvs28, Pvs25, partially orcompletely deglycosylated Pvs28, and Pvs28 fusion proteins, and thenucleic acids encoding such polypeptides, further comprisingnon-malarial sequences. For example, a Pvs25, Pvs28, or Pvs25/Pvs28polypeptide of the invention can further comprise epitope tags, enzymecleavage sequences, leader sequences, sequences which cause thepolypeptides to be transported to a particular intracellular organelle,and the like. For example, as discussed below, inclusion of yeast alphamating pheromone signal sequence in a fusion protein of the inventionallows for secretion of the expressed Pvs25 or Pvs28. These fusionproteins can provide for simplified manufacturing of Pvs25-Pvs28antigens.

In one class of embodiments, the Pvs25-Pvs28 fusion protein includes anN terminal Pvs25 domain and a C terminal Pvs28 domain. This arrangementof Pvs25 and Pvs28 in a fusion protein provides superior antigenic andtransmission blocking properties for the fusion protein. In onepreferred embodiment, the C terminal Pvs28 domain includes the carboxylterminal region of Pvs28. Exemplar fusion proteins are provided in theexamples set forth herein, and conservative modifications thereof.

Typically, the Pvs25-Pvs28 fusion proteins of the invention include aflexible linker separating the Pvs25 and Pvs28 domains. An exemplarflexible linker is the amino acid sequence GGGPGGG (SEQ ID NO:15).

In one embodiment, the fusion proteins (as Pvs25 and Pvs28) are producedrecombinantly. The recombinant proteins of the invention can beexpressed, e.g., in vitro, in prokaryotic or in eukaryotic systems. Inalternative embodiments, bacterial, yeast, insect, plant, mammalian, orother expression systems can be used.

In another embodiment, a nucleic acid encoding a fusion protein of theinvention is optimized for expression in a particular expression system,such as preferred codon usage in bacteria or partial or completedeglycosylation by mutation for yeast expression, thereby facilitatingrecombinant expression and manufacturing of the polypeptide of theinvention. For example, Pvs25 and Pvs28 consist of four epidermal growthfactor-like (EGF) domains (similar domains are found in the relatedPfs25 and Pfs28 Plasmodium polypeptides). These EGF domains comprisestructural domains in the molecules. In alternative embodiments, theimmunogen includes one or more domains in a variety of permutations andorientations. As domains may require disulfide bonds to create andmaintain structural integrity, alternative embodiments encompass variousexpression systems that faithfully recreate these disulfide linkages.

In another embodiment, the Pvs25, Pvs28 or Pvs25-Pvs28 fusion proteinsequences can be mutated or altered, e.g., using site-specificmutational methodologies. For example, in one embodiment, the Pvs25 andPvs28 sequences are mutated to eliminate one, several or all potentialglycosylation sites. Such mutations can facilitate recombinantexpression and manufacturing of the polypeptides of the invention, as inyeast expression systems. The partially or completely deglycosylated Pvspolypeptides of the invention are, in some circumstances, betterimmunogens, i.e., administration of these forms enhance the antigenicityof the polypeptide. For example, in one embodiment, an amino acidresidue at position 130 of Pvs28 is altered to remove a potentialglycosylation site.

In other embodiments, the different domains of the immunogeniccomposition are joined, or linked, together by chemical means. Infurther embodiments, the domains of the immunogenic compositions arederived from natural sources.

The Pvs25-Pvs28 fusion protein, when administered to a mammal, elicitsthe production of at least two classes of antibodies: antibodies whichspecifically bind to Pvs25, and antibodies which specifically bind toPvs28. In preferred embodiments, the administration of the fusionproteins of the invention elicit a transmission blocking immuneresponse. Immunological enhancers and pharmaceutically acceptablecarriers are optionally added to the fusion protein to enhance theimmunogenicity of the fusion protein and to facilitate delivery of thefusion protein to a mammal. For example, in alternative embodiments,adjuvants such as alum are added.

Immunogenic compositions comprising the fusion proteins of the inventionelicit transmission blocking antibodies in a variety of mammals,including humans and other primates, and mice and other rodents.

Cells expressing the nucleic acids and polypeptide of the invention area feature of the invention. For example, recombinant cells such as yeastcells can be used to express the Pvs25-Pvs28 fusion protein of theinvention. Cell lines containing a nucleic acid encoding the immunogenicpolypeptides and fusion proteins in an expression vector are alsodisclosed.

The invention provides methods of inducing a transmission blockingantibody in a mammal. In the methods, the Pvs25-Pvs28 fusion protein, ora nucleic acid encoding the fusion protein is administered to a mammal,which produces transmission blocking antigens. Administration istypically performed intramuscularly, intradermally, or subcutaneously.An adjuvant such as alum is optionally administered with the fusionprotein or nucleic acid.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification, the figures and claims.

All publications, patents and patent applications cited herein arehereby expressly incorporated herein by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary polynucleotide sequence for a Pvs28 of theinvention (SEQ ID NO:1).

FIG. 2 shows an exemplary Pvs28 polypeptide of the invention (SEQ IDNO:2).

FIG. 3 shows an exemplary polynucleotide sequence for a Pvs25 of theinvention (SEQ ID NO:3).

FIG. 4 shows an exemplary Pvs25 polypeptide of the invention (SEQ IDNO:4).

FIG. 5 shows an exemplary Pvs28-Pvs25 fusion protein polypeptide of theinvention (SEQ ID NO:5). GGGPGGG linker sequence=SEQ ID NO:15.

FIG. 6 shows exemplary constructs and recombinant proteins encoded bythese constructs (Pvs25=SEQ ID NO:16; Pvs28=SEQ ID NO:17; Pvs28Q130=SEQID NO:18; Pvs28NCR=SEQ ID NO:19), including Pvs25, Pvs28, and partiallydeglycosylated Pvs28 (having a glutamine residue, rather thanasparagine, at amino acid residue number 130).

FIG. 7 is a schematic representation of the an exemplary protocol forimmunization of mice with recombinant forms of Pvs25 and Pvs28.

DEFINITIONS

To facilitate understanding the invention, a number of terms are definedbelow.

An “immunogen” refers to a compound or composition comprising a peptide,polypeptide or protein which is “immunogenic,” i.e., capable ofeliciting, augmenting or boosting a cellular and/or humoral immuneresponse, either alone or in combination or linked or fused to anothersubstance. An immunogenic composition can be a peptide of at least about5 amino acids, a peptide of 10 amino acids in length, a fragment 15amino acids in length, a fragment 20 amino acids in length or greater.The immunogen can comprise a “carrier” polypeptide and a hapten, e.g., afusion protein or a carrier polypeptide fused or linked (chemically orotherwise) to another composition (described below). The immunogen canbe recombinantly expressed in an immunization vector, which can besimply naked DNA comprising the immunogen's coding sequence operablylinked to a promoter, e.g., a simple expression cassette. The immunogenincludes antigenic determinants, or epitopes (described below), to whichantibodies or TCRs bind, which are typically 3 to 10 amino acids inlength.

The term “Pvs25” and “Pvs28” polynucleotide refers to nucleic acidmolecules which encode Pvs25 and Pvs28 polypeptides, respectively, andnucleotides with substantial identity to these sequences, as describedbelow. Pvs25 and Pvs28 polypeptides are polypeptides containing asequence identical to or substantially identical (defined below) to theamino acid sequence of a class of 28 kD proteins expressed on thesurface Plasmodium vivax ookinetes. An exemplary polynucleotide sequencefor a Pvs28 of the invention is shown in SEQ ID NO:1, FIG. 1. Anexemplary amino acid sequence for a Pvs28 polypeptide of the inventionis shown in SEQ ID NO:2, FIG. 2. An exemplary polynucleotide sequencefor a Pvs25 of the invention is shown in SEQ ID NO:3, FIG. 3. Anexemplary amino acid sequence for a Pvs25 polypeptide of the inventionis shown in SEQ ID NO:4, FIG. 4. The term “Pvs25” and “Pvs28polypeptide” encompasses native proteins as well as recombinantlyproduced modified proteins that induce an immune response, including atransmission blocking immune response. It also includes immunologicallyactive fragments of these proteins. The terms “Pvs25” and “Pvs28polypeptide” also encompasses partially or completely deglycosylatedforms. A Pvs25 and Pvs28 polypeptide of the invention can be full lengthor an immunologically active fragment. The polypeptides will typicallybe between about 30 and 200 amino acids, typically at least about 100amino acids in length. Typically Pvs25 and Pvs28 polypeptides arecharacterized by their ability to induce transmission blocking immuneresponses, alone, or, as Pvs25/Pvs28 fusion proteins. The term “Pvs25”and “Pvs28 polypeptide” encompasses homologues and allelic variants ofPvs28 or Pvs25. Such homologues, also referred to as Pvs28 or Pvs25polypeptides, respectively, include variants of the native proteinsconstructed by in vitro techniques, and proteins from parasites relatedto P. vivax and P. falciparum. For example, one skilled in the art willappreciate that for certain uses it is advantageous to produce a Pvs28or Pvs25 polypeptide sequence that is lacking a structuralcharacteristic; e.g., one may remove a transmembrane domain to obtain apolypeptide that is more soluble in aqueous solution. The Pvs25 andPvs28 polypeptides of the invention, and sequences encoding theseproteins, also include fusion proteins comprising non-malarialsequences, e.g., epitope tags, enzyme cleavage recognition sequences,signal sequences, secretion signals (e.g., yeast alpha mating pheromonesignal sequence) and the like.

In the expression of recombinant genes, such as expression cassette orvector-expressed sequences or transgenes, one of skill will recognizethat the inserted polynucleotide sequence need not be identical and maybe “substantially identical” to a sequence of the gene from which it wasderived. As explained below, these variants are specifically covered bythe term Pvs25 and Pvs28. These variations include partially orcompletely deglycosylated forms of the polypeptides, and the nucleicacids which encode these variations.

In the case where the inserted polynucleotide sequence is transcribedand translated to produce a functional polypeptide, one of skill willrecognize that because of codon degeneracy a number of polynucleotidesequences will encode the same polypeptide. These variants arespecifically covered by the above term. In addition, the term“polynucleotide sequence from a Pvs25 (or Pvs28) gene” specificallyincludes those sequences substantially identical (determined asdescribed below) with a Pvs25 or Pvs28 gene sequence and that encodeproteins that retain the function of the Pvs25 or Pvs28 protein,respectively. Thus, in the case of the Pvs25 and Pvs28 gene disclosedherein, the above term includes variant polynucleotide sequences whichhave substantial identity with the sequences disclosed here and whichencode proteins capable of inducing an immune response, such as, but notlimited to, a transmission blocking immune response.

A “fusion protein” refers to a composition comprising at least onepolypeptide or peptide domain which is associated with a second domain.The second domain can be a polypeptide, peptide, polysaccharide, or thelike. The “fusion” can be an association generated by a peptide bond, achemical linking, a charge interaction (e.g., electrostatic attractions,such as salt bridges, H-bonding, etc.) or the like. If the polypeptidesare recombinant, the “fusion protein” can be translated from a commonmessage. Alternatively, the compositions of the domains can be linked byany chemical or electrostatic means. The Pvs25 and Pvs28 fusion proteinsof the invention can also include non-malarial sequences, e.g., linkers,epitope tags, enzyme cleavage recognition sequences, signal sequences,secretion signals, and the like.

A “Pvs25-Pvs28 fusion protein” refers to a polypeptide comprising atleast two domains, with polypeptide subsequences derived from both Pvs25and Pvs28. The fusion protein, in alternative embodiments, typicallyincludes about 10 contiguous amino acids or more; 15 contiguous aminoacids or more; 20 contiguous amino acids or more; and 25 contiguousamino acids or more from both Pvs25 and Pvs28. The Pvs25-Pvs28 fusionprotein can comprise additional subsequences which are not derived fromPvs25 or Pvs28, such as a flexible linker region separating the Pvs25and Pvs28 subsequences, epitope tags, etc., as discussed above.

An “N terminal” or “C terminal” domain in reference to a specifiedprotein refers to a polypeptide subsequence derived from the N terminalor C terminal half of the indicated protein. For example, an N terminalPvs25 protein domain refers to a polypeptide subsequence derived fromthe N terminal half of the Pvs25 protein. Similarly, a C terminal Pvs28protein domain refers to a polypeptide subsequence derived from the Cterminal half of the Pvs28 protein. The subsequence is from about 10amino acids in length up to the entire specified half protein.

The term “subsequence” in the context of a particular nucleic acidsequence or polypeptide refers to a region of the nucleic acid orpolypeptide equal to or smaller than the specified nucleic acid orpolypeptide.

A “recombinant nucleic acid” comprises or is encoded by one or morenucleic acids that are derived from a nucleic acid which wasartificially constructed. For example, the nucleic acid can comprise orbe encoded by a cloned nucleic acid formed by joining heterologousnucleic acids as taught, e.g., in Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger) and in Sambrook et al. (1989) MolecularCloning—A Laboratory Manual (2nd ed.) Vol. 1-3 (Sambrook).Alternatively, the nucleic acid can be synthesized chemically. The term“recombinant” when used with reference to a cell indicates that the cellreplicates or expresses a nucleic acid, or expresses a peptide orprotein encoded by a nucleic acid whose origin is exogenous to the cell.Recombinant cells can express genes that are not found within the native(non-recombinant) form of the cell. Recombinant cells can also expressgenes found in the native form of the cell wherein the genes arere-introduced into the cell or a progenitor of the cell by artificialmeans.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The term “complementary to” is used herein to mean thatthe complementary sequence is identical to all or a portion of areference polynucleotide sequence.

The nucleic acid and polypeptide sequences of the invention includesgene and protein products, respectively, identified and characterized byanalysis of Pvs 25 and Pvs28 sequences of the nucleic acids and proteinsof the invention. Optimal alignment of sequences for comparison can useany means to analyze sequence identity (homology) known in the art,e.g., by the progressive alignment method of termed “PILEUP” (seebelow); by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2: 482 (1981); by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson (1988) Proc. Natl. Acad. Sci. USA 85: 2444; by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.); ClustalW (CLUSTAL in the PC/Gene program byIntelligenetics, Mountain View, Calif., described by Higgins (1988)Gene, 73: 237-244; Corpet (1988) Nucleic Acids Res. 16:10881-90; Huang(1992) Computer Applications in the Biosciences 8:155-65, and Pearson(1994) Methods in Molec. Biol. 24:307-31), TreeAlign, MALIGN, and SAMsequence alignment computer programs; or, by inspection. See alsoMorrison (1997) Mol. Biol. Evol. 14:428-441, as an example of the use ofPILEUP. PILEUP, creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The methodused is similar to the method described by Higgins & Sharp (1989) CABIOS5: 151-153. The program can align up to 300 sequences of a maximumlength of 5,000. The multiple alignment procedure begins with thepairwise alignment of the two most similar sequences, producing acluster of two aligned sequences. This cluster can then be aligned tothe next most related sequence or cluster of aligned sequences. Twoclusters of sequences can be aligned by a simple extension of thepairwise alignment of two individual sequences. The final alignment isachieved by a series of progressive, pairwise alignments. The programcan also be used to plot a dendogram or tree representation ofclustering relationships. The program is run by designating specificsequences and their amino acid or nucleotide coordinates for regions ofsequence comparison.

Another example of algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul (1990)J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation, available on the worldwide web at ncbi.nlm.nih.gov/; seealso Zhang (1997) Genome Res. 7:649-656 (1997) for the “PowerBLAST”variation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence that either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased. Extensionof the word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a wordlength (W) of 11,the BLOSUM62 scoringmatrix (see Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparisonof both strands. The BLAST algorithm performs a statistical analysis ofthe similarity between two sequences (see, e.g., Karlin (1993) Proc.Natl. Acad. Sci. USA 90: 5873-5787). One measure of similarity providedby the BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i. e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 80% sequenceidentity, preferably at least 85%, more preferably at least 90% and mostpreferably at least 95%, compared to a reference sequence, the programsdescribed above using standard parameters. Thus, if a sequence has about80% sequence homology to a known Psv25 or Pvs28 polynucleotide orvariant thereof, then that sequence is considered a specie of Pvs25 orPvs28, respectfully. One of skill will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning andthe like.

“Substantial identity” of amino acid sequences for these purposes meanssequence identity of at least 40%, preferably at least 60%, morepreferably at least 90%, and most preferably at least 95%. Thus, if asequence has about 40% sequence homology to a known Psv25 or Pvs28polypeptide or variant thereof, then that sequence is considered aspecie of Psv25 or Pvs28, respectfully. Polypeptides which are“substantially similar” share sequences as noted above except thatresidue positions which are not identical may differ by conservativeamino acid changes. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, asp artic acid-glutamic acid, and asparagine-glutamine.

Determination of “substantial identity” can be focused over definedsubsequences, such as known structural domains. For example, for Pfs25and Pvs28, another measure of structural similarity will be the strikingalignment of cysteine (cys) residues and the spacing between the cysresidues. The reason why these residues are of higher importance thanothers is that they are critically involved in recreating the disulfidebond arrangements that comprise the EGF-like domains. These domains arethe hallmarks of Pvs25 and Pvs28, as with the related Plasmodiumpolypeptides Pfs25 and Pfs28.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other, or a third nucleic acid,under stringent conditions. Stringent conditions are sequence dependentand will be different in different circumstances. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (Tm) for the specific sequence at a defined ionic strength and pH.The Tm is the temperature (under defined ionic strength and pH) at which50% of the target sequence hybridizes to a perfectly matched probe.Typically, stringent conditions will be those in which the saltconcentration is about 1 molar at pH 7 and the temperature is at leastabout 60° C.

In the present invention, mRNA encoded by the nucleic acids of theinvention can be identified in Northern blots under stringent conditionsusing the sequences disclosed here or fragments of, typically, at leastabout 100 nucleotides. For the purposes of this disclosure, stringentconditions for such RNA-DNA hybridizations are those which include atleast one wash in 6×SSC for 20 minutes at a temperature of at leastabout 50° C., usually about 55° C. to about 60° C., or equivalentconditions.

Another indication that protein sequences are substantially identical isif one protein is immunologically reactive with antibodies raisedagainst the other protein. Thus, the proteins of the invention includeproteins immunologically reactive with antibodies raised against Pvs 25and Pvs28 polypeptides, and fusion proteins thereof.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given polypeptide.For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation. One of skill will recognize that each codonin a nucleic acid (except AUG, which is ordinarily the only codon formethionine, and UGG, the single codon for Trp) can be modified to yielda functionally identical molecule by standard techniques. Accordingly,each “silent variation” of a nucleic acid which encodes a polypeptide isimplicit in each described sequence.

The term “conservatively modified variations” refers to individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (typically lessthan 5%, more typically less than 1%) in an encoded sequence, where thealterations result in the substitution of an amino acid with achemically similar amino acid; and the alterations, deletions oradditions do not alter the structure, function and/or immunogenicity ofthe sequence. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A “vector” is a composition which can transduce, transfect, transform orinfect a cell, thereby causing the cell to replicate or express nucleicacids and/or proteins other than those native to the cell, or in amanner not native to the cell. A cell is “transduced” by a nucleic acidwhen the nucleic acid is translocated into the cell from theextracellular environment. Any method of transferring a nucleic acidinto the cell may be used; the term, unless otherwise indicated, doesnot imply any particular method of delivering a nucleic acid into acell, nor that any particular cell type is the subject of transduction.A cell is “transformed” by a nucleic acid when the nucleic acid istransduced into the cell and stably replicated. A vector includes anucleic acid (ordinarily RNA or DNA) to be expressed by the cell. Thisnucleic acid is optionally referred to as a “vector nucleic acid.” Avector optionally includes materials to aid in achieving entry of thenucleic acid into the cell, such as a viral particle, liposome, proteincoating or the like. A “cell transduction vector” is a vector whichencodes a nucleic acid which is expressed in a cell once the nucleicacid is transduced into the cell.

A “promoter” is an array of nucleic acid control sequences which directtranscription of a nucleic acid. As used herein, a promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elementswhich can be located as much as several thousand base pairs from thestart site of transcription. A “constitutive” promoter is a promoterwhich is active under most environmental and developmental conditions.An “inducible” promoter is a promoter which is under environmental ordevelopmental regulation. A “tissue specific” promoter is active incertain tissue types of an organism, but not in other tissue types fromthe same organism. The term “operably linked” refers to a functionallinkage between a nucleic acid expression control sequence (such as apromoter, or array of transcription factor binding sites) and a secondnucleic acid sequence, wherein the expression control sequence directstranscription of the nucleic acid corresponding to the second sequence.

A “susceptible organism” is a Plasmodium host that is susceptible tomalaria, for example, humans and chickens. The particular susceptibleorganism or host will depend upon the Plasmodium species.

As used herein, “isolated,” when referring to a molecule or composition,such as, e.g., a Pvs25 or Pvs28 nucleic acid or polypeptide, means thatthe molecule or composition is separated from at least one othercompound, such as a protein, other nucleic acids (e.g., RNAs), or othercontaminants with which it is associated in vivo or in its naturallyoccurring state. Thus, a Pvs25 or Pvs28 composition is consideredisolated when the Pvs25 or Pvs28 has been isolated from any othercomponent with which it is naturally associated, e.g., cell membrane, asin a cell extract. An isolated composition can, however, also besubstantially pure. An isolated composition can be in a homogeneousstate and can be in a dry or an aqueous solution. Purity and homogeneitycan be determined, for example, using analytical chemistry techniquessuch as polyacrylamide gel electrophoresis (SDS-PAGE) or highperformance liquid chromatography (HPLC). Thus, the isolated Pvs25 orPvs28 compositions of this invention do not contain materials normallyassociated with their in situ environment. Even where a protein has beenisolated to a homogenous or dominant band, there are trace contaminantswhich co-purify with the desired protein.

A “transmission blocking antibody” is an antibody which inhibits thegrowth or replication of a malarial parasite during the sexual stage ofparasite development in the mosquito gut. The term “antibody” refers toa polypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as myriad immunoglobulinvariable region genes. Light chains are classified as either kappa orlambda. Heavy chains are classified as gamma, mu, alpha, delta, orepsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,IgD and IgE, respectively. An exemplar immunoglobulin (antibody)structural unit comprises a tetramer. Each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus ofeach chain defines a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. The terms variablelight chain (V_(L)) and variable heavy chain (V_(H)) refer to theselight and heavy chains respectively. Antibodies exist e.g., as intactimmunoglobulins or as a number of well characterized fragments producedby digestion with various peptidases. Thus, for example, pepsin digestsan antibody below the disulfide linkages in the hinge region to produceF(ab)′₂, a dimer of Fab which itself is a light chain joined toV_(H)-C_(H)l by a disulfide bond. The F(ab)′₂ may be reduced under mildconditions to break the disulfide linkage in the hinge region therebyconverting the F(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer isessentially an Fab with part of the hinge region (see, FundamentalImmunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. (1993),for a more detailed description of other antibody fragments). Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such Fab′ fragmentsmay be synthesized de novo either chemically or by utilizing recombinantDNA methodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies. Immunoglobulins generated using recombinant expressionlibraries are also antibodies for purposes of this invention.

An “immunogenic composition” is a composition which elicits theproduction of antibodies or a cell-mediated immune response whenadministered to a mammal.

An “immunological carrier” or “carrier” in the immunological context (asopposed to a carrier which is a nonactive composition for the purpose offormulating, storing or carrying a pharmaceutical) is an compositionwhich, when linked, joined, chemically coupled or fused to a secondcomposition (e.g., protein, peptide, polysaccharide or the like) boostsor augments the cellular or humoral response to the composition. Anyphysiologic mechanism can be involved in this augmentation or boostingof the immune response. An immunogenic carrier is typically apolypeptide linked or fused to a second composition of interestcomprising a protein, peptide or polysaccharide, where the carrierstimulates a cellular (T cell mediated) immune response that boosts oraugments the humoral (B cell mediated, antibody-generating) immuneresponse to the composition of interest. These second compositions canbe “haptens,” which are typically defined as compounds of low molecularweight that are not immunogenic by themselves, but that, when coupled tocarrier molecules, can elicit antibodies directed to epitopes on thehapten. For example, the lack of an adequate immune response to themajor polysaccharide of the Haemophilus influenzae type b capsule (PRP)in very young infants can be overcome by conjugating PRP to a T-celldependent carrier protein (see Zepp (1997) Eur. J. Pediatr. 156:18-24).Alternatively, a peptide can be linked to a carrier simply to facilitatemanipulation of the peptide in the generation of the immune response(see, e.g., Rondard (1997) Biochemistry 36:8962-8968).

An “epitope” refers to an antigenic determinant or antigen site thatinteracts with an antibody or a T cell receptor (TCR). An “antigen” is amolecule or composition that induces the production of an immuneresponse. An antibody or TCR binds to a specific conformational(possibly charge-dependent) domain of the antigen, called the “antigenicdeterminant” or “epitope” (TCRs bind the epitope in association with athird molecule, a major histocompatibility complex (MHC) protein).

DETAILED DESCRIPTION

The present invention relates to novel compositions and methods forblocking transmission of malaria, particularly Plasmodium vivax. Theinvention provides agents which are capable eliciting antibodies andantiserum (generated by administration of the compositions of theinvention) which, when ingested by the mosquito, are capable ofinhibiting the life cycle of the disease-causing parasite in themosquito midgut. The agents include Plasmodium vivax Pvs 25 and Pvs28polypeptides (including partially and completely deglycosylated forms),nucleic acids encoding these polypeptides, and fusion proteins thereof,that are useful for inducing antibodies that block transmission of theparasite. The invention also provides the isolated antibodies generatedby these polypeptides. These nucleic acid and polypeptide compositionsare useful as vaccines against malaria.

This invention further relates to an immunogenic composition capable ofeliciting an immunogenic response directed to an epitope comprising anisolated Pvs25 or Pvs28 and an isolated molecule comprising the epitope.The invention is also directed to methods of eliciting an immunogenicresponse directed to an epitope comprising administering an isolatedPvs28 and an isolated molecule comprising the epitope. In oneembodiment, the Pvs28 is acting as an immunogenic carrier to a haptenepitope to elicit, stimulate or augment a humoral immune response to theepitope.

The fusion proteins of the invention (optionally used with an adjuvantsuch as alum) can be used to block transmission of a number of parasitesassociated with malaria. Examples of parasites whose transmission isblocked by the materials and compositions of the invention include thecausative parasites for malaria. Four species of the genus Plasmodiuminfect humans, P. vivax, P. ovale, P. malariae, and P. falciparum. P.falciparum is the most prevalent cause of malaria in humans: OtherPlasmodium species infect other animals. For instance, P. gallinaceum isresponsible for avian malaria.

The present invention relates to recombinant viruses and vaccinescomprising nucleic acid sequences which encode malaria parasite(Plasmodium vivax) Pvs25 and Pvs28 polypeptides, including fusionproteins and deglycosylated forms (SEQ ID NOs: 1 to 5). Thesepolypeptides are naturally expressed by Plasmodium during itsmosquito-infective, sexual stage. Because naturally expressed Pvspolypeptides are expressed in malaria parasite oocytes and zygotes,recombinant forms can be used to induce an immune response to the sexualstage of the parasite.

These Pvs25- and Pvs28-expressing malaria parasite sexual stages occuronly in the mosquito host and not in the human. This invention includescompositions and methods for eliciting human antibodies which, wheningested by the mosquito during its feeding process, block thedevelopment of malaria in the mosquito. Blocking the sexual developmentof the malaria parasite in the mosquito reduces the vector's ability tofurther transmit the disease to a second human host.

The human antiserum generated by the compositions and methods of theinvention, when ingested by the mosquito, significantly reduces thenumbers of malaria parasite oocysts within the insect. Significantpublic health benefits are attained by the vaccines' ability to elicitantibodies which, upon mosquito ingestion, significantly decrease thenumber of oocysts capable of maturing into infectious sporozoites. Avaccine is still very useful when it generates an antiserum thatdecreases the numbers of oocysts in the mosquito, thus reducing thenumbers of parasites transmitted by the mosquito. To be useful, it isnot necessary that the ingested antisera render the mosquito completelyincapable of transmitting the malaria parasite to a second person (i.e.,completely inhibit sexual development of all oocysts).

The use of sexual stage polypeptides as a transmission blocking antigensare described, e.g., in U.S. Pat. No. 5,217,898 to Kaslow and Barrdirected to Pfs25 as a transmission blocking antigen, and U.S. Pat. No.5,527,700 to Kaslow and Duffy, directed to Pfs28 as a transmissionblocking antigen.

The Pvs25-Pvs28 fusion proteins of the invention have several surprisingproperties. The fusion protein is more efficient in producingtransmission blocking antibodies, e.g., in mice, than Pvs25 or Pvs28alone. This is true despite the fact a mixed dose of Pvs25 and Pvs28will not induce a higher level of transmission blocking antibodyactivity than either Pvs25 or Pvs28 alone. Second, less fusion proteinis required as an immunogen than either Pvs25 or Pvs28 alone. Third,titers of transmission blocking antibodies will remain high for a longerperiod of time when the antigen is a Pvs25-Pvs28 fusion protein thaneither Pvs25 or Pvs28 alone. In a preferred aspect, the inventionprovides a nucleic acid with yeast preferred codons for encoding andexpressing the fusion protein in yeast.

Pvs28 and Pvs25 Polypeptides

The present invention includes immunogenic Pvs 25 and Pvs28 polypeptidesand fragments derived from these proteins, and partially or completelydeglycosylated forms of these polypeptides, that are useful for inducingan immune response when the proteins are injected into a human or otherhost animal. An exemplary polynucleotide sequence for a Pvs25 of theinvention is shown in SEQ ID NO:3, FIG. 3. An exemplary amino acidsequence for a Pvs25 polypeptide of the invention is shown in SEQ IDNO:4, FIG. 4. An exemplary polynucleotide sequence for a Pvs28 of theinvention is shown in SEQ ID NO:1, FIG. 1. An exemplary amino acidsequence for a Pvs28 polypeptide of the invention is shown in SEQ IDNO:2, FIG. 2.

In another embodiment, the immunogenic composition, comprising anisolated Pvs28 and an isolated molecule comprising the epitope, iscapable of eliciting or augmenting an immunogenic response directed tothe epitope. The Pvs28 can act as a immunological “carrier” to boost,augment or increase the cellular or humoral response to the epitope. Theantibodies that arise from the immune response block transmission of theparasite by interfering with the portion of the parasite's life cyclethat occurs in the mosquito. For example, purified polypeptides havingan amino acid sequence substantially identical to a subsequence of Pvs28may be used; including partially or completely deglycosylated forms ofPvs28.

The antibodies or T cells that arise from administration of Pvs28, Pvs25or Pvs28-Pvs25 fusion proteins (e.g., as in a polypeptide vaccine, or avaccine comprising nucleic acid encoding these polypeptides, such as avirus or vector) generate an immune response by blocking transmission ofthe parasite malaria by interfering with the portion of the parasite'slife cycle that occurs in the mosquito. Pvs 25 and Pvs28 are similar instructure to other known ookinete antigens such as Pfs25 and Pfs28,respectively. All four proteins comprise a putative secretory signalsequence, followed by four EGF-like domains and a terminal hydrophobictransmembrane region without a cytoplasmic tail. Although the fourproteins share the six-cysteine motif of the EGF-like domains, thefunctions of these proteins may be very different. EGF-like domains havebeen recognized in a range of proteins that have diverse functions(Davis (1990) New Biol. 2:410-419).

Included among the polypeptides of the present invention are proteinsthat are variants of the native proteins constructed by in vitro or invivo techniques, including recombinant or synthetic techniques. Oneskilled in the art will appreciate, for instance, that for certain usesit would be advantageous to produce a Pvs25 or a Pvs28 polypeptide thatis lacking one of its structural characteristics. For example, one mayremove the transmembrane domain to obtain a polypeptide that is moresoluble in aqueous solution.

Alternatively, the invention provides partially and completelydeglycosylated variants, such as the genetically engineered Pvs28 of theinvention in which the amino acid at position 130 does not encode anasparagine, and thus cannot be a putative site for N-linkedglycosylation. In an exemplary sequence, the nucleic acid of theinvention was modified to encode glutamine, and the Pvs28 variantpolypeptide of the invention was modified to be glutamine at residue130. However, any putative amino acid site of N— or O-linkedglycosylation (and the nucleic acid which encodes such a site, or motif)can be modified to alternatively be (or encode, in the case of thenucleic acid) any amino acid residue incapable of acting as aglycosylation signal.

The Pvs28 and Pvs25 proteins of the invention may be purified fromparasites isolated from infected host organisms. For a review ofstandard techniques see, e.g., Methods in Enzymology, “Guide to ProteinPurification”, M. Deutscher, ed. Vol. 182 (1990); Scopes, R. K., ProteinPurification: Principles and Practice, 2nd ed., Springer Verlag, (1987).For instance, Pvs25 and Pvs28 polypeptides can be purified usingaffinity chromatography, SDS-PAGE, and the like. Illustrative examplesof methods for purifying Pvs25, Pvs28 and fusion proteins thereof of theinvention are described below. Methods for purifying desired proteinsare well known in the art and are not presented in detail here.

Solubility Fractionation

If the protein mixture is complex, an initial salt fractionation canseparate many of the unwanted host cell proteins (or proteins derivedfrom the cell culture media) from the recombinant protein of interest.The preferred salt is ammonium sulfate. Ammonium sulfate precipitatesproteins by effectively reducing the amount of water in the proteinmixture. Proteins then precipitate on the basis of their solubility. Themore hydrophobic a protein is, the more likely it is to precipitate atlower ammonium sulfate concentrations. A typical protocol is to addsaturated ammonium sulfate to a protein solution so that the resultantammonium sulfate concentration is between 20-30%. This will precipitatethe most hydrophobic of proteins. The precipitate is discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

If the size of the protein of interest is known or can be estimated fromthe cDNA sequence, proteins of greater and lesser size can be removed byultrafiltration through membranes of different pore size (for example,Amicon or Millipore membranes). As a first step, the protein mixture isultrafiltered through a membrane with a pore size that has a lowermolecular weight cut-off than the molecular weight of the protein ofinterest. The retentate of the ultrafiltration is then ultrafilteredagainst a membrane with a molecular cut off greater than the molecularweight of the protein of interest. The recombinant protein will passthrough the membrane into the filtrate. The filtrate can then bechromatographed.

Column Chromatography

Proteins can be separated on the basis of their size, net surfacecharge, hydrophobicity and affinity for ligands. In addition, antibodiesraised against proteins can be conjugated to column matrices and theproteins immunopurified. All of these general methods are well known inthe art. See Scopes (1987) supra. Chromatographic techniques can beperformed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech). Protein concentrations can bedetermined using any technique, e.g., as in Bradford (1976) Anal.Biochem. 72:248-257.

Amino Acid Sequence Determination

Illustrative amino acid sequences of the Pvs28 and Pvs25 and fusionproteins of this invention can be determined by, for example, Edmandegradation, a technique which is well known in the art. In addition tothe internal sequencing (see also Hwang (1996) J. Chromatogr. B. Biomed.Appl. 686:165-175), N-terminal sequencing can be performed by techniquesknown in the art. For C-terminal sequence determination, a chemicalprocedure for the degradation of peptides and analysis bymatrix-assisted-laser-desorption ionization mass spectrometry (MALDI-MS)can be used Thiede (1997) Eur. J. Biochem. 244:750-754.

Molecular Weight/Isoelectric Point Determination

The molecular weight of a protein can be determined by many differentmethods, all known to one of skill in the art. Some methods ofdetermination include: SDS gel electrophoresis, native gelelectrophoresis, molecular exclusion chromatography, zonalcentrifugation, mass spectroscopy, and calculation from sequencing.Disparity between results of different techniques can be due to factorsinherent in the technique. For example, native gel electrophoresis,molecular exclusion chromatography and zonal centrifugation depend onthe size of the protein. The proteins that are cysteine rich can formmany disulfide bonds, both intra- and intermolecular. SDS gelelectrophoresis depends on the binding of SDS to amino acids present inthe protein. Some amino acids bind SDS more tightly than others,therefore, proteins will migrate differently depending on their aminoacid composition. Mass spectroscopy and calculated molecular weight fromthe sequence in part depend upon the frequency that particular aminoacids are present in the protein and the molecular weight of theparticular amino acid. If a protein is glycosylated, mass spectroscopyresults will reflect the glycosylation but a calculated molecular weightmay not.

The isoelectric point of a protein can be determined by native gel (ordisc) electrophoresis, isoelectric focussing or in a preferred method,by calculation given the amino acid content of the protein (see, forexample, Wehr (1996) Methods Enzymol. 270:358-374; Moorhouse (1995) J.Chromatogr. A. 717:61-69, describing capillary isoelectric focusing).

Pvs25-Pvs28 Fusion Proteins

The present invention includes immunogenic polypeptides which comprisepolypeptide subsequences derived from both Pvs28 and Pvs25, includingthe exemplary fusion protein of the invention SEQ ID NO:5 (see FIG. 5)and deglycosylated forms. These polypeptides are useful for inducing animmune response when the fusion protein is injected into a human, mouseor other host animal. The antibodies that arise from the immune responseblock transmission of the malarial parasite by interfering with theportion of the parasite's life cycle that occurs in the mosquito.

The fusion proteins typically include an immunogenic domain, or epitope,from a Pvs25 and an immunogenic domain, or epitope from a Pvs28(including deglycosylated forms). The immunogenic domains, or epitopes,are peptide and polypeptide subsequences of the correspondingpolypeptides which are sufficient to elicit an immunogenic response(antibody or T cell response) against the domain when administered to amammal (e.g., a mouse or a human). In one embodiment, the immunogenicdomain can elicit the production of an antibody which recognizes thecorresponding full length protein. For example, if the immunogenicdomain is a Pvs25 subsequence, the domain (epitope) elicits theproduction of an antibody which specifically binds to Pvs25. Similarly,if the immunogenic domain is a Pvs28 subsequence, the domain preferablyelicits the production of an antibody which specifically binds to Pvs28.

To elicit the production of an antibody, the immunogenic domain istypically at least about 3-10 amino acids in length, because the proteinrecognition site on an antibody typically recognizes an amino acid ofabout 3-10 amino acids in length. More often, the immunogenic domain islonger than 10 amino acids, and the domain optionally includes the fulllength sequence of the corresponding protein (i.e., in one embodiment,the Pvs25-Pvs28 fusion protein comprises the complete sequence of bothPvs25 and Pvs28). Ordinarily, only a fraction of the full length proteinis included. In one embodiment, about 10% of the full length Pvs25 isincluded in the fusion protein. In another embodiment, about 20% of thefull length Pvs25 is included in the fusion protein. In yet anotherembodiment, about 30% of the full length protein is included. In stillanother embodiment, about 40% of the full length Pvs25 is included inthe fusion protein. Optionally, as much as about 50% of the full lengthPvs25 is included in the fusion protein. Occasionally, as much as about60% of the full length Pvs25 is included in the fusion protein. In someembodiments, as much as about 70% of the full length Pvs25 is includedin the fusion protein. In one class of embodiments, as much as about 80%of the full length Pvs25 is included in the fusion protein. As much asabout 90% of the full length Pvs25 is optionally included in the fusionprotein. As already mentioned, the entire full length Pvs25 protein isoptionally incorporated into the fusion protein.

Similarly, in one embodiment, about 10% of the full length Pvs28isincluded in the fusion protein. In another embodiment, about 20% of thefull length Pvs28 is included in the fusion protein. In yet anotherembodiment, about 30% of the full length protein is included. In stillanother embodiment, about 40% of the full length Pvs28 is included inthe fusion protein. Optionally, as much as about 50% of the full lengthPvs28 is included in the fusion protein. Occasionally, as much as about60% of the full length Pvs28 is included in the fusion protein. In someembodiments, as much as about 70% of the full length Pvs28 is includedin the fusion protein. In one class of embodiments, as much as about 80%of the full length Pvs28 is included in the fusion protein. As much asabout 90% of the full length Pvs28 is optionally included in the fusionprotein. As already mentioned, the entire full length Pvs28 protein isoptionally incorporated into the fusion protein.

The portion of the Pvs25 or Pvs28 protein from which the immunogenicdomain, or epitope, is selected is optionally optimized for maximumimmunogenicity for the induction of transmission blocking vaccines. Anycombination of Pvs25 and Pvs28 subsequences (epitopes) can be combined.Any combination of complete or partially deglycosylated subsequences canbe combined. In alternative embodiments, the Pvs25 and Pvs28 epitopescan be in alternating or sequential patterns. For example, in oneembodiment, the carboxyl terminal portion of Pvs28 is included.Embodiments also include those derived from fusion proteins in whichabout 10-20 amino acids are deleted or added to the particular Pvs25 orPvs28 subsequences described. The added or deleted amino acids are addedor deleted by reference to the corresponding full length sequence, e.g.,where the subsequence is derived from Pvs25, a 10-20 amino acid sequencederived from Pvs25 is optionally added to either end of the subsequence.

The fusion proteins optionally includes additional features such as aflexible linker between Pvs25 and Pvs28 domains. The linkers canfacilitate the independent folding of the Pvs25 and Pvs28 proteins.Preferred flexible linkers are amino acid subsequences which aresynthesized as part of a recombinant fusion protein. In one embodiment,the flexible linker is an amino acid subsequence comprising a prolinesuch as Gly₃-Pro-Gly₃ (SEQ ID NO:15). In other embodiments, a chemicallinker is used to connect synthetically or recombinantly produced Pvs25and Pvs28 subsequences. Such flexible linkers are known to persons ofskill in the art. For example, poly(ethylene glycol) linkers areavailable from Shearwater Polymers, Inc. Huntsville, Ala. These linkersoptionally have amide linkages, sulfhydryl linkages, or heterofunctionallinkages.

In addition to flexible linkers, the fusion proteins optionally includepolypeptide subsequences from proteins which are unrelated to Pvs25 orPvs28, e.g., a sequence with affinity to a known antibody to facilitateaffinity purification, detection, or the like. Such detection- andpurification-facilitating domains include, but are not limited to, metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between thepurification domain and Pvs25 or Pvs28 protein(s) may be useful tofacilitate purification. One such expression vector provides forexpression of a fusion protein comprising the sequence encoding a Pvs25or Pvs28 of the invention, or a fusion protein thereof, and nucleic acidsequence encoding six histidine residues followed by thioredoxin and anenterokinase cleavage site (for example, see Williams (1995)Biochemistry 34:1787-1797). The histidine residues facilitate detectionand purification while the enterokinase cleavage site provides a meansfor purifying the desired protein(s) from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the patent andscientific literature, see e.g., Kroll (1993) DNA Cell. Biol.,12:441-53).

An exemplary fusion of Pvs25 to Pvs28 by a flexible linker isrepresented by the polypeptide of FIG. 5, SEQ ID NO:5, whose individualdomains are:

a Pvs25 sequence (with or without a signal sequence or anchor):

AVTVDTICKNGQLVQMSNHFKCMCNEGLVHLSENTC (SEQ ID NO:20)EEKNECKKETLGKACGEFGQCIENPDPAQVNMYKCGCIEGYTLKEDTCVLDVCQYKNCGESGECIVEYLSEIQSAGCSCAIGKVPNPEDEKKCTKTGETACQLKCNTD NEVCKNVEGVYKCQCMEGFTFDKEKNVCLS;

with a flexible linker, e.g.: GGGPGGG (SEQ ID NO:15); and

a Pvs28 sequence (with or without signal sequence or anchor):

AKVTAETQCKNGYVVQMSNHFECKCNDGFVMANENT (SEQ ID NO:21)CEEKRDCTNPQNVNKNCGDYAVCANTRMNDEERALRCGCILGYTVMNEVCTPNKCNGVLCGKGKCILDPANVNSTMCSCNIGTTLDESKKCGKPGKTECTLKCKANEECKETQNYYKCVAKGSGGEGSGGEGSGGEGSGGEGSG GEGSGGDTGAAYSLMN.

The fusion protein (and a Pvs25 or Pvs28 polypeptide) can also include asecretory signal sequence, e.g., in mammalian cell expression: Igsecretion signal or tPA signal sequence; or a pre-pro secretion signal,e.g., in yeast: alpha-factor.

Included among the polypeptides of the present invention are fusionproteins that have subsequences which are homologues or allelic variantsof Pvs28 or Pvs25. Such homologues, also referred to as Pvs28 or Pvs25polypeptides, respectively, include variants of the native proteinsconstructed by in vitro techniques, and proteins from parasites relatedto P. vivax and P. falciparum. For example, one skilled in the art willappreciate that for certain uses it is advantageous to produce a Pvs28or Pvs25 polypeptide subsequence that is lacking a structuralcharacteristic; e.g., one may remove a transmembrane domain (to obtain apolypeptide that is more soluble in aqueous solution) or a glycosylationsite (to obtain a polpeptide that is more antigenic under certainconditions).

One of skill will appreciate that many conservative variations of thefusion proteins and nucleic acid which encode the fusion proteins yieldessentially identical products. For example, due to the degeneracy ofthe genetic code, “silent substitutions” (i.e., substitutions of anucleic acid sequence which do not result in an alteration in an encodedpolypeptide) are an implied feature of every nucleic acid sequence whichencodes an amino acid. As described herein, sequences are preferablyoptimized for expression in a particular host cell used to produce thefusion protein (e.g., yeast). Similarly, “conservative amino acidsubstitutions,” in one or a few amino acids in an amino acid sequenceare substituted with different amino acids with highly similarproperties (see, the definitions section, supra), are also readilyidentified as being highly similar to a particular amino acid sequence,or to a particular nucleic acid sequence which encodes an amino acid.Such conservatively substituted variations of any particular sequenceare a feature of the present invention.

One of skill will recognize many ways of generating alterations in agiven nucleic acid sequence, which optionally provides alterations to anencoded protein. Such well-known methods include site-directedmutagenesis, PCR amplification using degenerate oligonucleotides,exposure of cells containing the nucleic acid to mutagenic agents orradiation, chemical synthesis of a desired oligonucleotide (e.g., inconjunction with ligation and/or cloning to generate large nucleicacids) and other well-known techniques. See, Giliman and Smith (1979)Gene 8:81-97; Roberts et al. (1987) Nature 328:731-734 and Sambrook,Innis, Ausbel, Berger, Needham VanDevanter and Mullis (below).

Most commonly, amino acid sequences are altered by altering thecorresponding nucleic acid sequence and expressing the polypeptide.However, polypeptide sequences are also optionally generatedsynthetically on commercially available peptide synthesizers to produceany desired polypeptide (see, Merrifield, and Stewart and Young, supra).

One can select a desired nucleic acid or polypeptide of the inventionbased upon the sequences and constructs provided and upon knowledge inthe art regarding malaria generally. The life-cycle, genomicorganization, developmental regulation and associated molecular biologyof malaria strains have been the focus of research since the advent ofmolecular biology.

Moreover, general knowledge regarding the nature of proteins and nucleicacids allows one of skill to select appropriate sequences with activitysimilar or equivalent to the nucleic acids, vectors and polypeptidesdisclosed herein. The definitions section herein describes exemplarconservative amino acid substitutions.

Finally, most modifications to nucleic acids and polypeptides areevaluated by routine screening techniques in suitable assays for thedesired characteristic. For instance, changes in the immunologicalcharacter of a polypeptide can be detected by an appropriateimmunological assay. Modifications of other properties such as nucleicacid hybridization to a target nucleic acid, redox or thermal stabilityof a protein, hydrophobicity, susceptibility to proteolysis, or thetendency to aggregate are all assayed according to standard techniques.

Pvs25 Pvs28 and Pvs25-Pvs28 Nucleic Acids

Another aspect of the present invention relates to the cloning andrecombinant expression (using expression cassettes, plasmids, vectors,recombinant viruses, and the like) of Pvs 25 and Pv28 proteins, variants(i.e. deglycosylated forms) construction of Pvs25-Pvs28 fusion proteins,as described above. The recombinantly expressed proteins can be used ina number of ways. For instance, they can be used astransmission-blocking vaccines or as immunogens to raise antibodies, asdescribed below. In addition, oligonucleotides from the cloned genes canbe used as probes to identify homologous, allelic and variant species ofPvs polypeptides in Plasmodium vivax, Plasmodium sp., and in otherspecies.

Thus, the invention relies on routine techniques in the field ofrecombinant genetics, well known to those of ordinary skill in the artand well described in the scientific and patent literature, e.g., basictexts disclosing the general methods of use in this invention includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Publish., Cold Spring Harbor, NY 2nd ed. (1989) (Sambrook); andCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, Greene Publishing Associates, Inc. and John Wiley &Sons, Inc. (1995 Supplement). Product information from manufacturers ofbiological reagents and experimental equipment also provide informationuseful in known biological methods. Such manufacturers include, e.g.,the SIGMA chemical company (Saint Louis, MO), R&D systems (Minneapolis,Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., AldrichChemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL LifeTechnologies, Inc. (Gaithersberg, Md.), Fluka Chemica-BiochemikaAnalytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen, San Diego,Calif., and Applied Biosystems (Foster City, Calif.), as well as manyother commercial sources known to one of skill.

In summary, the manipulations necessary to prepare nucleic acid segmentsencoding the polypeptides and introduce them into appropriate host cellsinvolve 1) purifying the polypeptide from the appropriate sources, 2)preparing degenerate oligonucleotide probes corresponding to a portionof the amino acid sequence of the purified proteins, 3) screening a cDNAor genomic library for the sequences which hybridize to the probes, 4)constructing vectors comprising the sequences linked to a promoter andother sequences necessary for expression and 5) inserting the vectorsinto suitable host cells or viruses.

After isolation of the desired protein as described above, the aminoacid sequence of the N-terminus is determined and degenerateoligonucleotide probes, designed to hybridize to the desired gene, aresynthesized. Amino acid sequencing is performed and oligonucleotideprobes are synthesized according to standard techniques as described,e.g., in Sambrook or Ausubel.

Genomic or cDNA libraries are prepared according to standard techniquesas described, e.g., in Sambrook or Ausubel. To construct genomiclibraries, large segments of genomic DNA are generated by randomfragmentation and are ligated with vector DNA to form concatemers thatcan be packaged into the appropriate vector. Two kinds of vectors arecommonly used for this purpose, bacteriophage lambda vectors andplasmids.

To prepare cDNA, mRNA from the parasite of interest is first isolated.Eukaryotic mRNA has at its 3′ end a string of adenine nucleotideresidues known as the poly-A tail. Short chains of oligo d-T nucleotidesare then hybridized with the poly-A tails and serve as a primer for theenzyme, reverse transcriptase. This enzyme uses RNA as a template tosynthesize a complementary DNA (cDNA) strand. A second DNA strand isthen synthesized using the first cDNA strand as a template. Linkers areadded to the double-stranded cDNA for insertion into a plasmid or phagevector for propagation in E. coli.

cDNA can also be prepared using PCR (see below for further discussionPCR). PCR is used to produce high-quality cDNA from nanograms of totalor poly A+ RNA. For example, the CapFinder™ PCR cDNA Synthesis Kit(Clonetech, Palo Alto, Calif.) was used to identify and isolate cDNAfrom Plasmodium. This technique utilizes long-distance PCR (Barnes(1994) Proc. Natl. Acad. Sci. USA 91:2216-2220, Cheng (1994) Proc. Natl.Acad. Sci. USA 91:5695-5699) to generate high yields of representative,double-stranded cDNA. See also, e.g., Zhu (July 1996) CLONTECHniquesXI(3):12-13; CLONTECHniques (October 1995) X(4):2-5; and CLONTECHniques(January 1996) XI(1):2-4.

Identification of clones in either genomic or cDNA libraries harboringthe desired nucleic acid segments is performed by either nucleic acidhybridization or immunological detection of the encoded protein, if anexpression vector is used. The bacterial colonies are then replicaplated on solid support, such as nitrocellulose filters. The cells arelysed and probed with either oligonucleotide probes described above orwith antibodies to the desired protein.

Other methods well known to those skilled in the art are used toidentify desired genes, i.e., various species of Pvs25 and Pvs28 of theinvention. For example, the presence of restriction fragment lengthpolymorphisms (RFLP) between wild type and mutant strains lacking aPvs25 or Pvs28 polypeptide can be used.

Oligonucleotides can be used to identify and detect Pvs25 and Pvs28using a variety of hybridization techniques and conditions. For example,amplification techniques, such as the polymerase chain reaction (PCR)can be used to amplify the desired nucleotide sequence. One of skill inthe art will appreciate that, whatever amplification method is used, ifa quantitative result is desired, care must be taken to use a methodthat maintains or controls for the relative frequencies of the amplifiednucleic acids. Suitable amplification methods include, but are notlimited to: polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TOMETHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCRSTRATEGIES (1995), ed. Innis, Academic Press, Inc., NY (“Innis ”); and,U.S. Pat. Nos. 4,683,195 and 4,683,202 describe this method), ligasechain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science241:1077; Barringer (1990) Gene 89:117); transcription amplification(Kwoh Proc. Natl. Acad. Sci. USA, 86:1173 (1989)); and, self-sustainedsequence replication (Guatelli (1990) Proc. Natl. Acad. Sci. USA,87:1874); Q Beta replicase amplification (Smith (1997) J. Clin.Microbiol. 35:1477-1491, automated Q-beta replicase amplification assay;Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316, Sambrook, and Ausubel,as well as Mullis (1987) U.S. Pat. Nos. 4,683,195 and 4,683,202; Arnheim(1990) C&EN 36-47; Lomell J. Clin. Chem., 35:1826 (1989); Van Brunt,Biotechnology, 8:291-294 (1990); Wu (1989) Gene 4:560; Sooknanan (1995)Biotechnology 13:563-564. Methods for cloning in vitro amplified nucleicacids are described in Wallace, U.S. Pat. No. 5,426,039.

The invention provides for amplification and manipulation or detectionof the products from each of the above methods to prepare DNA encoding aPvs25 or Pvs28 protein specie. In PCR techniques, oligonucleotideprimers complementary to the two borders of the DNA region to beamplified are synthesized an used (see, e.g., Innis). PCR can be used ina variety of protocols to amplify, identify, isolate and manipulatenucleic acids encoding Pvs25 or Pvs 28. In these protocols, appropriateprimers and probes for identifying and amplifying DNA encoding Pvs25 orPvs 28 polypeptides and fragments thereof are generated that compriseall or a portion of any of the DNA sequences listed herein.PCR-amplified sequences can also be labeled and used as detectableoligonucleotide probes, but such nucleic acid probes can be generatedusing any synthetic or other technique well known in the art, asdescribed above. The labeled amplified DNA or other oligonucleotide ornucleic acid of the invention can be used as probes to further identifyand isolate Pvs25 or Pvs 28 protein isoforms or alleles or Pvs25 or Pvs28 from various cDNA or genomic libraries.

The present invention also provides RACE-based methods for isolatingPvs25 or Pvs 28 nucleic acids from any organism (RACE is anotherPCR-based approach for DNA amplification). Briefly, this techniqueinvolves using PCR to amplify a DNA sequence using a random 5′ primerand a defined 3′ primer (5′ RACE) or a random 3′ primer and a defined 5′primer (3′ RACE). The amplified sequence is then subcloned into a vectorwhere can be sequenced and manipulated using standard techniques. TheRACE method is well known to those of skill in the art and kits toperform RACE are commercially available, e.g. Gibco BRL, Gaithersburg,Md., #18374-058 (5′ RACE) or #18373-019 (3′ RACE), see also Lankiewicz(1997) Nucleic Acids Res 25:2037-2038; Frohman (1988) Proc. Natl. Acad.Sci. USA 85:8998; Doenecke (1997) Leukemia 11:1787-1792.

For 5′ RACE, a primer, the gene-specific primer, is selected near the 5′end of the known sequence oriented to extend towards the 5′ end. Theprimer is used in a primer extension reaction using a reversetranscriptase and mRNA. After the RNA is optionally removed, thespecifically-primed cDNA is either: 1) “tailed” with deoxynucleotidetriphosphates (dNTP) and dideoxyterminal transferase, then a primer thatis complimentary to the tail with a 5′ end that provides a unique PCRsite and the first gene-specific primer is used to PCR amplify the cDNA.Subsequent amplifications are usually performed with a gene-specificprimer nested with respect to the first primer, or 2) an oligonucleotidethat provides a unique PCR site is ligated to an end of the cDNA usingRNA ligase; then a primer complimentary to the added site and the firstgene-specific primer is used to PCR amplify the cDNA, with subsequentamplifications usually performed with a gene-specific primer nested withrespect to the first primer. Amplified products are then purified,usually by gel electrophoresis then sequenced and examined to seecontain the additional cDNA sequences desired.

For 3′ RACE, an oligo dT-primer is annealed to the poly-A tails of anmRNA and then extended by a reverse transcriptase. Usually the oligo dTprimer has a 5′ end that provides a unique PCR site. The RNA is thenremoved, optionally, or dissociated, and the cDNA is amplified with aprimer to the oligo dT tail and a gene-specific primer near the 3′ endof the known sequence (oriented towards the 3′ end). Subsequentamplifications are usually performed with a gene-specific primer nestedwith respect to the first primer. Amplified products are then purified,usually by gel electrophoresis then sequenced and examined to seecontain the additional cDNA sequences desired.

Sequences amplified by PCR can be purified from agarose gels and clonedinto an appropriate vector according to standard techniques.

Standard transfection methods are used to produce prokaryotic,mammalian, yeast or insect cell lines which express large quantities ofthe Pvs25 or Pvs 28 polypeptide, which is then purified using standardtechniques, as described above. See, e.g., Colley (1989) J. Biol. Chem.264:17619-17622; and Scopes, supra.

The polypeptides of the present invention can be readily designed andmanufactured utilizing various recombinant DNA or synthetic techniqueswell known to those skilled in the art. For example, the polypeptidescan vary from the naturally-occurring sequence at the primary structurelevel by amino acid, insertions, substitutions, deletions, and the like.These modifications can be used in a number of combinations to producethe final modified protein chain.

The amino acid sequence variants can be prepared with various objectivesin mind, including immunogenicity, facilitating purification, andpreparation of the recombinant polypeptide. Design of completely orpartially deglycosylated polypeptides improve the antigenicity of theimmunogenic composition, as discussed above. Modified polypeptides canalso be useful for modifying plasma half life, improving therapeuticefficacy, and lessening the severity or occurrence of side effectsduring therapeutic use. The amino acid sequence variants are usuallypredetermined variants not found in nature but exhibit the same, orimproved (in the case of deglycosylation variants) immunogenic activityas naturally occurring, Pvs25 and Pvs28 polypeptides. For instance,polypeptide fragments comprising only a portion (usually at least about60-80%, typically 90-95%) of the primary structure may be produced. Foruse as vaccines, polypeptide fragments are typically preferred so longas at least one epitope capable of eliciting transmission blockingantibodies remains. In the construction of deglycosylation variants,amino acid motifs which act as N-linked or O-linked glycosylationsignals (which are well known in the art, see, e.g., Kakinuma (1997) JBiol Chem 272:28296-28300) are modified to forms (motif variants) thatare not recognized as glycosylation sites in the expression systems inwhich the recombinant form is produced.

The nucleotide sequences used to express the polypeptides of theinvention and to transfect the host cells can be modified according tostandard techniques to yield Pvs25-Pvs28, Pvs25 or Pvs28 polypeptides,fusion proteins, variants or fragments thereof, with a variety ofdesired properties. For example, the invention also provides for Pvs25and Pvs28 which have been modified in a site-specific manner to modifyor delete any or all functions or epitopes. Site-specific mutations canbe introduced into Pvs25 and Pvs28-encoding nucleic acid by a variety ofconventional techniques, well described in the scientific and patentliterature. For example, one rapid method to perform site-directedmutagenesis efficiently is the overlap extension polymerase chainreaction (OE-PCR) (Urban (1997) Nucleic Acids Res. 25 :2227-2228). Otherillustrative examples include: site-directed mutagenesis by overlapextension polymerase chain reaction (OE-PCR), as in Urban (1997) NucleicAcids Res. 25:2227-2228; Ke (1997) Nucleic Acids Res 25:3371-3372, andChattopadhyay (1997) Biotechniques 22:1054-1056, describing PCR-basedsite-directed mutagenesis “megaprimer” method; Bohnsack (1997) Mol.Biotechnol. 7:181-188; Ailenberg (1997) Biotechniques 22:624-626,describing site-directed mutagenesis using a PCR-based staggeredre-annealing method without restriction enzymes; Nicolas (1997)Biotechniques 22:430-434, site-directed mutagenesis using longprimer-unique site elimination and exonuclease III. See Gillman (1979)Gene 8:81-97; Roberts (1987) Nature 328:731-734.

In general, modifications of the sequences encoding the homologouspolypeptides may be readily accomplished by a variety of well-knowntechniques, such as site-directed mutagenesis, described above. One ofordinary skill will appreciate that the effect of many mutations isdifficult to predict. Thus, most modifications are evaluated by routinescreening in a suitable assay for the desired characteristic. Forinstance, the effect of various modifications on the ability of thepolypeptide to elicit transmission blocking can be easily determinedusing the mosquito feeding assays, described below. In addition, changesin the immunological character of the polypeptide can be detected by anappropriate competitive binding assay. Modifications of other propertiessuch as redox or thermal stability, hydrophobicity, susceptibility toproteolysis, or the tendency to aggregate are all assayed according tostandard techniques.

The particular procedure used to introduce the genetic material into thehost cell for expression of the Pvs 25 and Pvs28 polypeptide is notparticularly critical. Any of the well known procedures for introducingforeign nucleotide sequences into host cells may be used. These includethe use of calcium phosphate transfection, spheroplasts,electroporation, liposomes, microinjection, plasma vectors, viralvectors and any of the other well known methods for introducing clonedgenomic DNA, cDNA, synthetic DNA or other foreign genetic material intoa host cell (see, e.g., Sambrook, Ausubel, supra). It is only necessarythat the particular procedure utilized be capable of successfullyintroducing at least one gene into the host cell which is capable ofexpressing the gene.

The particular vector used to transport the genetic information into thecell is also not particularly critical. Any of the conventional vectorsused for expression of recombinant proteins in prokaryotic andeukaryotic cells may be used.

Expression vectors for mammalian cells typically contain regulatoryelements from eukaryotic viruses. SV40 vectors include pSVT7 and pMT2.Vectors derived from bovine papilloma virus include pBV-1MTHA, andvectors derived from Epstein Bar virus include pHEBO, and p2O5.

Other exemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5,bacculovirus pDSVE, and any other vector allowing expression of proteinsunder the direction of the SV-40 early promoter, SV-40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

The expression vector typically contains a transcription unit orexpression cassette that contains all the elements required for theexpression of the Pvs28 or Pvs25 polypeptide DNA in the host cells. Atypical expression cassette contains a promoter operably linked to theDNA sequence encoding a Pvs28 or Pvs25 polypeptide and signals requiredfor efficient polyadenylation of the transcript. The term “operablylinked” as used herein refers to linkage of a promoter upstream from aDNA sequence such that the promoter mediates transcription of the DNAsequence. The promoter is preferably positioned about the same distancefrom the heterologous transcription start site as it is from thetranscription start site in its natural setting. As is known in the art,however, some variation in this distance can be accommodated withoutloss of promoter function.

The DNA sequence encoding the Pvs28 or Pvs25 polypeptide will typicallybe linked to a cleavable signal peptide sequence to promote secretion ofthe encoded protein by the transformed cell. Additional elements of thecassette may include selectable markers, enhancers and, if genomic DNAis used as the structural gene, introns with functional splice donor andacceptor sites.

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream from the transcription initiation site. Many enhancerelements derived from viruses have a broad host range and are active ina variety of tissues. For example, the SV40 early gene enhancer issuitable for many cell types. Other enhancer/promoter combinations thatare suitable for the present invention include those derived frompolyoma virus, human or murine cytomegalovirus, the long term repeatfrom various retroviruses such as murine leukemia virus, murine or Roussarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, ColdSpring Harbor Pres, Cold Spring Harbor, N.Y. 1983.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

If the mRNA encoded by the structural gene is to be efficientlytranslated, polyadenylation sequences are also commonly added to thevector construct. Two distinct sequence elements are required foraccurate and efficient polyadenylation: GU or U rich sequences locateddownstream from the polyadenylation site and a highly conserved sequenceof six nucleotides, AAUAAA, located 11-30 nucleotides upstream.Termination and polyadenylation signals that are suitable for thepresent invention include those derived from SV40, or a partial genomiccopy of a gene already resident on the expression vector.

Pvs25 or Pvs28 coding sequences can be inserted into a host cell genomebecoming an integral part of the host chromosomal DNA, using forexample, retroviral vectors such as SIV or HIV, see for example, Naldini(1996) Science 272:263-267; Vanin (1997) J. Virol. 71:7820-7826;Zufferey (1997) Nat. Biotechnol. 15:871-875, describing attenuatedlentiviral vector gene delivery in vivo; Feng (1997) Nat. Biotechnol.15:866-870, describing stable in vivo gene transduction viaadenoviral/retroviral chimeric vector.

Nucleic acids of the invention can used in DNA immunization techniques.Coding sequence is operably linked to expression cassettes or vectorsand injected directly as “naked” DNA into the host. The DNA can beinjected intramuscularly or intradermally. See. e.g., Donnelly (1995)Ann. NY Acad. Sci. 772:40-46; Corr (1997) J. Immunol. 159:4999-5004;Manickan (1997) J. Clin. Invest. 100:2371-2375. Variations of thistechnique use cationic liposome-entrapped DNA vaccines (see Gregoriadis(1997) FEBS Lett. 402:107-110); immunization with naked plasmid DNAtransfected in dendritic cells (Manickan (1997) J. Leukoc. Biol.61:125-132); and, cutaneous genetic immunization with naked DNA (Condon(1996) Nat. Med. 2:1122-1128).

Yeast expression systems, being eukaryotic, provide an attractivealternative to bacterial systems for some applications; for an overviewof yeast expression systems, see. e.g., Protein Engineering Principlesand Practice, eds. Cleland et al., Wiley-Liss, Inc. p 129 (1996), Barr(1988) J. Biol. Chem. 263: 16471-16478, or U.S. Pat. No. 4,546,082. Avariety of yeast vectors are publicly available. For example, theexpression vector pPICZ B (Invitrogen, San Diego, Calif.) has beenmodified to create expression vectors of the invention to express thePvs25 or Pvs28 of the invention in yeast, such as S. cerevisiase andPichia pastoris. Yeast episomal plasmids comprising inducible promoterscan be used for the intracellular expression of the Pvs25 or Pvs28proteins of the invention. Vectors include the pYES2 expression vector(Invitrogen, San Diego, Calif.) and pBS24.1 (Boeke (1984) Mol. Gen.Genet. 197:345); see also Jacobs (1988) Gene 67:259-269.

One embodiment uses the yeast expression vector comprising theRecombinant Protein Expression Unit called YEpRPEU-1, -2 and -3; andpIXY154 (Immunex Corp.). pIXY154 and YEpRPEU-3 have been used to expressPvs25, Pvs28 and Pvs28-Q130, amutagenized form of Pvs28 which eliminatesall, several, or, one potential N-linked glycosylation site, asdiscussed herein.

Yeast promoters for yeast expression vectors suitable for the expressionof a Pvs25 or Pvs28 include the inducible promoter from the alcoholdehydrogenase gene, ADH2, also called the yeast alcohol dehydrogenase IIgene promoter (ADH2P) (La Grange (1997) Appl. Microbiol. Biotechnol.47:262-266). In one embodiment, the ADH2 promoter is modified to includea tract of poly A to enhance the ADH2 promoter in the expression of thepolypeptides of the invention. Suitable promoters to use also includethe ADH2/GAPDH hybrid promoter as described, e.g., in Cousens (1987)Gene 61:265-275.

In another embodiment, the Pvs25 or Pvs28 to be expressed can also befused at the amino terminal end to the secretion signal sequence of theyeast mating pheromone alpha-factor (MF alpha 1S) and fused at thecarboxy terminal end to the alcohol dehydrogenase II gene terminator(ADH2T), see van Rensburg (1997) J. Biotechnol. 55:43-53. The yeastalpha mating pheromone signal sequence allows for secretion of theexpressed Pvs25 or Pvs28. In one embodiment, sequences are added afterthe KEX-2 cleavage site to enhance cleavage of the alpha factor leader;preferred embodiments include addition of the sequence EAEA (SEQ IDNO:22) and EAEAEAEAK (SEQ ID NO:23).

Yeast cell lines suitable for the present invention include e.g., BJ2168 (Berkeley Yeast Stock Center) as well as other commonly availablelines. For example, the yeast can be a Pichia sp., Hansenula sp.,Torulopsis sp., Saccharomyces sp., or a Candida sp. The yeast canspecifically be a Pichia pastoris, Hansenula polymorpha, Torulopsisholmil, Saccharomyces fragilis, Saccharomyces cerevisiae, Saccharomyceslactis, or a Candida pseudotropicalis. In other embodiments,Saccharomyces cerevisiae cell lines XV2181 from Immunex; and, 2905/6,VQ1 and VK1 which we have developed as our own yeast expression hosts.

Any of a number of other well known cells and cell lines can be used toexpress the polypeptides of the invention. For instance, prokaryoticcells such as E. coli can be used. Eukaryotic cells include, Chinesehamster ovary (CHO) cells, COS cells, mouse L cells, mouse A9 cells,baby hamster kidney cells, C127 cells, PC8 cells, and insect cells.

Following the growth of the recombinant cells and expression of thePvs25 or Pvs28 polypeptide, the culture medium is harvested forpurification of the secreted protein. The media are typically clarifiedby centrifugation or filtration to remove cells and cell debris and theproteins are concentrated by adsorption to any suitable resin such as,for example, CDP-Sepharose, Asialoprothrombin-Sepharose 4B, or QSepharose, or by use of ammonium sulfate fractionation, polyethyleneglycol precipitation, or by ultrafiltration. Other routine means knownin the art may be equally suitable. Further purification of the Pvs25 orPvs28 or fusion polypeptide can be accomplished by standard techniques,for example, affinity chromatography, metal affinity chromatography(IMAC) (see, e.g., Govoroun (1997) J. Chromatogr. B. Biomed. Sci. Appl.698:35-46; Froelich (1996) Biochem. Biophys. Res. Commun. 229:44-49),ion exchange chromatography, sizing chromatography or other proteinpurification techniques to obtain homogeneity, as described above. Thepurified proteins are then used to produce pharmaceutical compositions,as described below.

Transmission-Blocking Antibodies

A further aspect of the invention includes antibodies against Pvs25 orPvs28 polypeptides. The antibodies are useful for diagnostic purposes orfor blocking transmission of parasites. The antibodies of the inventionmay be polyclonal or monoclonal. Typically, polyclonal sera arepreferred.

Antibodies are typically tetramers of immunoglobulin polypeptides. Asused herein, the term “antibody” refers to a protein consisting of oneor more polypeptides substantially encoded by immunoglobulin genes.Immunoglobulin genes include those coding for the light chains, whichmay be of the kappa or lambda types, and those coding for the heavychains. Heavy chain types are alpha, gamma, delta, epsilon and mu. Thecarboxy terminal portions of immunoglobulin heavy and light chains areconstant regions, while the amino terminal portions are encoded by themyriad immunoglobulin variable region genes. The variable regions of animmunoglobulin are the portions that provide antigen recognitionspecificity. The immunoglobulins may exist in a variety of formsincluding, for example, Fv, Fab, and F(ab)₂, as well as in singlechains, e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883(1988) and Bird et al., Science 242: 423-426, 1988. See, generally, Hoodet al., Immunology, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller(1986) Nature, 323:15-16. Single-chain antibodies, in which genes for aheavy chain and a light chain are combined into a single codingsequence, may also be used.

Use of the Polypeptides or Nucleic Acids of the Invention to InduceImmune Responses.

The immunoglobulins, nucleic acids, and polypeptides of the presentinvention are also useful as prophylactics, or vaccines, for blockingtransmission of malaria or other diseases caused by parasites.Compositions containing the immunoglobulins, polypeptides, nucleic acidsor a cocktail thereof are administered to a subject, giving rise to ananti-Pvs25 or anti-Pvs28 polypeptide immune response in the mammalentailing the production of anti-Pvs25 or anti-Pvs28 polypeptideimmunoglobulins. The Pvs25 or Pvs28 polypeptide-specific immunoglobulinsthen block transmission of the parasite from the subject to thearthropod vector, preventing the parasite from completing its lifecycle. An amount of prophylactic composition sufficient to result in atiter of antiserum which, upon ingestion by the mosquito, is capable ofblocking transmission or is capable of decreasing ability of the oocyteto mature in the mosquito (resulting in fewer infective particles passedto the mosquitoes' next target bloodmeal), is defined to be an“immunologically effective dose.”

The isolated nucleic acid sequences coding for Pvs25 or Pvs28polypeptides can be used in viruses to transfect host cells in thesusceptible organism, particularly, a human. Live attenuated viruses,such as vaccinia or adenovirus, are convenient alternatives for vaccinesbecause they are inexpensive to produce and are easily transported andadministered. Vaccinia vectors and methods useful in immunizationprotocols are well known in the art and are described, e.g., in U.S.Pat. No. 4,722,848.

Suitable viruses for use in the present invention include, but are notlimited to, pox viruses, such as, canarypox and cowpox viruses, andvaccinia viruses, alpha viruses, adenoviruses, and other animal viruses.The recombinant viruses can be produced by methods well known in theart: for example, using homologous recombination or ligating twoplasmids together. A recombinant canarypox or cowpox virus can be made,for example, by inserting the gene encoding the Pvs25 or Pvs28, or otherhomologous polypeptide into a plasmid so that it is flanked with viralsequences on both sides. The gene is then inserted into the virus genomethrough homologous recombination.

A recombinant adenovirus virus can be produced, for example, by ligatingtwo plasmids each containing 50% of the viral sequence and the DNAsequence encoding the Pvs25 or Pvs28 polypeptide. Recombinant RNAviruses such as the alpha virus can be made via a cDNA intermediateusing methods known in the art.

The recombinant virus of the present invention can be used to induceanti-Pvs25 or anti-Pvs28 polypeptide antibodies in mammals, such as miceor humans. In addition, the recombinant virus can be used to produce thePvs25 or Pvs28 polypeptides by infecting host cells which in turnexpress the polypeptide.

The nucleic acids can also be used to produce other recombinantmicroorganisms such as bacteria, yeast, and the like. For instance, BCG(Bacille Calmette Guerin) vectors are described, e.g., in Stover (1991)Nature 351:456-460. A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g., Salmonella typhi, Saccharomyces vectors and the like,will be apparent to those skilled in the art from the descriptionherein.

The DNA encoding the polypeptides of the invention can also beadministered to the patient. Typically, an expression cassette suitablefor driving expression in human cells is prepared. This approach isdescribed, for instance, in Wolff (1990) Science 247:1465-1468; U.S.Pat. Nos. 5,580,859 and 5,589,466.

The present invention also relates to host cells infected with therecombinant virus of the present invention. The host cells of thepresent invention are preferably eukaryotic, such as yeast cells, ormammalian, such as BSC-1 cells. Host cells infected with the recombinantvirus express the Pvs25 or Pvs28 polypeptides on their cell surfaces. Inaddition, membrane extracts of the infected cells induce transmissionblocking antibodies when used to inoculate or boost previouslyinoculated mammals.

In the case of vaccinia virus (e.g., strain WR), the sequence encodingthe Pvs25 or Pvs28 polypeptides can be inserted into the viral genome bya number of methods including homologous recombination using a transfervector, pTKgpt-OFIS as described in Kaslow et al., Science252:1310-1313, 1991.

The Pvs25 or Pvs28 polypeptides or nucleic acids of the presentinvention can be used in pharmaceutical and vaccine compositions thatare useful for administration to mammals, particularly humans, to blocktransmission of a variety of infectious diseases. The compositions aresuitable for single administrations or a series of administrations. Whengiven as a series, inoculations subsequent to the initial administrationare given to boost the immune response and are typically referred to asbooster inoculations.

The pharmaceutical compositions of the invention are intended forparenteral, topical, oral or local administration. Preferably, thepharmaceutical compositions are administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration thatcomprise a solution of the agents described above dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier. A variety ofaqueous carriers may be used, e.g., phosphate buffered saline, water,buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.These compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient and more preferably at a concentration of 25%-75%.

For aerosol administration, the polypeptides or nucleic acids arepreferably supplied in finely divided form along with a surfactant andpropellant. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. A carrier can also be included, as desired,as with, e.g., lecithin for intranasal delivery.

In therapeutic applications, Pvs25 or Pvs28 polypeptides or nucleicacids of the invention are administered to a patient in an amountsufficient to prevent parasite development in the arthropod and thusblock transmission of the disease. An amount adequate to accomplish thisis defined as a “therapeutically effective dose.” Amounts effective forthis use will depend on, e.g., the particular polypeptide or virus, themanner of administration, the weight and general state of health of thepatient, and the judgment of the prescribing physician.

The vaccines of the invention contain as an active ingredient animmunogenically effective amount of the Pvs25 or Pvs28 polypeptides,nucleic acids, or recombinant virus as described herein. Useful carriersare well known in the art, and include, e.g., thyroglobulin, albuminssuch as human serum albumin, tetanus toxoid, polyamino acids such aspoly(D-lysine:D-glutamic acid), influenza, hepatitis B virus coreprotein, hepatitis B virus recombinant vaccine and the like. Thevaccines can also contain a physiologically tolerable (acceptable)diluent such as water, phosphate buffered saline, or saline, and furthertypically include an adjuvant. Adjuvants such as incomplete Freund'sadjuvant, aluminum phosphate, aluminum hydroxide, or alum are materialswell known in the art.

Vaccine compositions containing the polypeptides or nucleic acids of theinvention are administered to a patient to elicit atransmission-blocking immune response against the antigen and thusprevent spread of the disease through the arthropod vector. Such anamount is defined as an “immunogenically effective dose.” In this use,the precise amounts again depend on the patient's state of health andweight, the mode of administration, and the nature of the formulation.

As noted above, the Pvs25 or Pvs28 polypeptides of this invention mayalso be used to make monoclonal antibodies. Such antibodies may beuseful as potential diagnostic or therapeutic agents. The polypeptidesthemselves may also find use as diagnostic reagents. For example, apolypeptide of the invention may be used to diagnose the presence ofantibodies against P. vivax in a patient. Alternatively, thepolypeptides can be used to determine the susceptibility of a particularindividual to a particular treatment regimen, and thus may be helpful inmodifying an existing treatment protocol or in determining a prognosisfor an infected individual.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. Those of skill will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially similar results.

Example 1 Cloning of Pvs25

The following example details one exemplary means to isolation of aspecies of Plasmodium Pvs25. To isolate Plasmodium vivax gene encodingPvs25, the gene sequences of the eight known proteins-Pfs25, Pgs25,Pys25, Pbs25, Pfs28, Pgs28, Pys21 and Pbs21 were aligned and theirsequence similarities analyzed, as described above.

A highly conserved nucleotide sequence in the first EGF-like domain wasidentified. This sequence was used to synthesize a degenerate PCRoligonucleotide. To prevent the re-amplification of Pvs28 gene,nucleotides were chosen that were not identical to the Pvs28 sequence. Asense primer (5′-GG(AT) TTT (CT)T(AG) (AG)(CT)T CA(AG) ATG AGT-3′) (SEQID NO:6) was constructed. Using this primer with a vector-specific M13universal primer (5′-GTA AAA CGA CGG CCA GT-3′) (SEQ ID NO:7), nucleicacid sequences were amplified form a P. vivax genomic library (a P.vivax (Sal1) genomic library: Sau3AI partial digest cloned into pUC18BamHI/BAP). The PCR reaction was: 94° C. for 10 min, then 30 cycles of94° C. for 30 seconds, 44° C. for 60 seconds and 72° C. for 2 min 30seconds, and finally 72° C. for 8 min.

Two different sizes of DNA fragments were amplified in this reaction.These PCR products were again amplified by using an internal degenerateprimer (sense primer: 5′-TCA (AG)AT GAG T(AG)(AG) (CT)CA TTT (AGT)GAATG-3′) (SEQ ID NO:8) with a vector specific M13 universal primer (sameas described above) at: 94° C. for 10 min, then 30 cycles of 94° C. for30 seconds, 44° C. for 30 seconds and 72° C. for 1 min, and finally 72°C. for 10 min. The resultant amplified DNA was purified and cloned intopCR2.1 (Invitrogen). Using plasmid-specific sequencing primers, eightindividual recombinant plasmid clones were completely sequenced (ABIPRISM 310 Genetic Analyzer; PE Applied Biosystems). This yielded apartial DNA sequence of Pvs25.

The complete nucleotide sequence for Pvs25, DNA was amplified by anested splinkerette PCR method (see Devon (1995) Nucleic Acids Res 23:1644-1645; Hengen (1995) Trends Biochem. Sci. 20:372-373) using pairs ofgene-specific and splinkerette-specific primers. For the first PCR-sensesplinkerette #1 primer: 5′-CGA ATC GTA ACC GTT CGT ACG AGA A-3′ (SEQ IDNO:9); and an antisense Pvs25 specific primer: 5′-GGA CAA GCA GGA TGATAA AG-3′ (SEQ ID NO: 10). For nested PCR, sense splinkerette #2internal primer: 5′-TCG TAC CAG AAT CGC TGT CCT CTC C-3′ (SEQ ID NO:11);and an anti-sense Pvs25 specific internal primer: 5′-AGC ACA CAA GTG TCTTCC TTC-3′ (SEQ ID NO:12). The template DNA was prepared by the ligationof splinkerettes with VspI digested genomic DNA obtained from P. vivaxSal1 strain. Primary PCR using these primers combined Hot Start (TaqGold DNA polymerase, PE Applied Biosystems), and, Touchdown PCR tocircumvent spurious priming during gene amplification (see Don (1991)Nucleic Acids Res. 19: 4008). PCR protocols were as follows:denaturation 94° C. for 10 min in the first cycle and 30 secondsthereafter; annealing, for 1 min at 60° C. initially, decreasing by 2°C. to 50° C. per cycle and 50° C. thereafter; extension, 72° C. for 2min (cycles 1-10), then 4 min (cycles 11-20) and finally 6 min (cycles21-30). In the primary PCR reaction 0.2 ul of ligation product wasamplified in 20 ul. Secondary PCR was performed using 0.3 ul primary PCRproduct as a template, and the PCR condition were as follows: 94° C. for10 min, then 10 cycles of 94° C. for 30 seconds, 50° C. for 60 secondand 72° C. for 2 min, then 10 cycles of 94° C. for 30 seconds, 50° C.for 60 seconds and 72° C. for 4 min, and then 10 cycles of 94° C. for 30seconds, 50° C. for 60 seconds and 72° C. for 6 min, and finally 72° C.for 4 min. After the nested PCR, two different sized DNA fragments wereobserved.

After the purification of the individual amplified DNA fragments, eachDNA fragment was cloned into pCR2.1 (Invitrogen) and by usingplasmid-specific sequencing primers, eight individual recombinantplasmid clones were completely sequenced (ABI PRISM 310 GeneticAnalyzer; PE Applied Biosystems). The full length open reading frame ofPvs25 gene sequence (SEQ ID NO:3) was obtained from these sequences, andthe polypeptide sequence (SEQ ID NO:4) encoded therein deduced (FIGS. 3and 4, respectively).

A further pair of gene specific PCR primers was designed andconstructed: the sense primer 5′-ACT TTC GTT TCA CAG CAC-3′ (SEQ IDNO:13); the anti-sense primer 5′-AAA GGA CAA GCA GGA TGA TA-3′ (SEQ IDNO:14). The primers were complementary (designed to hybridize) at eachend of the gene sequence to amplify the full length sequence of Pvs25.By using these primers, full length Pvs25 gene was amplified from P.vivax Sallgenomic library (as above). After the purification of thespecific DNA fragment, we directly sequenced the DNA fragment by usingPvs25-specific sequencing primers (ABI PRISM 310 Genetic Analyzer; PEApplied Biosystems).

Analysis of the amino acid sequence deduced from the 657 base pair (bp)single ORF of Pvs25 revealed a presumptive secretory signal sequence,followed by four EGF-like domains with a total of 22 cysteines, and ashort hydrophobic region at the carboxy-terminus. The sequence was notthat of the Pvs28 gene; furthermore, the presence of six rather thanfour cysteines in the fourth EGF-like domain (a hallmark of P25homologues rather than P28 homologues) confirmed that the sequenceobtained was that of Pvs25.

Example 2 Expression of Pvs25 in Yeast

For expression in yeast, a Pvs25 DNA fragment was obtained by PCRamplification, as described above. This Pvs25 subsequence was designedto lack the presumptive secretory signal andglycosylphosphatidylinositol lipid (GPI) anchor (see, e.g., Gowda (1997)J. Biol. Chem. 272:6428-6439) sequences. A polyhistidine tag sequencewas also spliced into the polypeptide coding sequence.

The resultant nucleic acid construct encoding a Pvs25 fusion protein(SEQ ID NO:16) was ligated into the NheI and ApaI restriction sites ofthe yeast shuttle vector, YepRPEU-3, as schematically represented inFIG. 6. Recombinant clones were electroporated into the host S.cerevisiae strain, VK1, and clones harboring the recombinant plasmidwere screened for their ability to secrete a His6 (SEQ ID NO:24) taggedprotein. A single high-producing colony was amplified in selectivegrowth media and was used to establish a cell bank for yeast expressedPvs25.

For these and all yeast studies described herein, fermentation procedurewas essentially as described by Kaslow (1994) Biotechnology 12:494-499.A 1 ml frozen seed lot was thawed and used to inoculate 500 ml ofexpansion medium (8% glucose, 1% yeast nitrogen base, 2% acid-hydrolyzedcasamino acids, 400 mg/L adenine sulfate, 400 mg/L uracil) in a Tunairbaffled shaker flask. The cells were grown overnight at 30° C. withshaking at 250 rpm for 20-40 hr. The overnight growth in expansionmedium was used to inoculate 3-3.5 L of fermentation media (0.5%glucose, 1% yeast extract, 1% yeast nitrogen base, 2% acid-hydrolyzedcasamino acids, 400 mg/L adenine sulfate, 400 mg/L uracil). TheBioflo-III fermentor was set to keep pH at 5.02, temperature at 25° C.and dissolved oxygen at or above 60% by agitation between 360 and 1000rpm. A glucose-rich nutrient medium (25% glucose, 1% yeast extract, 1%yeast nitrogen base, 2% acid-hydrolyzed casamino acids, 0.5 g/L adeninesulfate, 0.5 g/L uracil, 2.5 g/L MgSO₄) was fed continuously at a rateof 25 ml/hr for approximately 40 hr. 25% NH₄OH was fed to keep pH at5.02. When OD₆₀₀ of the culture reached 50 units, the carbon source wasswitched from glucose to 30% ethanol, 20% glycerol to induce proteinsecretion for 10-16 hr.

The culture supernatant was recovered by centrifugation andfilter-sterilized through a 0.45 μm cellulose acetate membrane(Nalgene). The sterile medium was concentrated to 350 mLs using anAmicon tangential ultrafiltration apparatus fitted with a YD 10 spiralhollow fiber filter (Amicon), and then continuously dialyzed with 1.5 L2× PBS pH 7.4. The retentate was incubated with Ni—NTA agarose withshaking at 4° C. overnight. After overnight incubation, the suspensionwas transferred to a column and the resin was washed sequentially with2× PBS pH 7.4, 2× PBS pH 6.8 and 1× PBS pH 6.4. The protein was elutedfrom the resin using 0.250 M NaAcetate pH 4.5 and analyzed by SDS-PAGE.Further purification was performed by size-exclusion chromatographyusing a Pharmacia Superdex-75 column to which 1× PBS pH 7.4 was appliedat a flow rate of 1 mL/min. One mL fractions were collected and analyzedby SDS-PAGE. Fractions containing the Pfs25 (and, in other experiment,the Pvs28, or the ˜39 kD fusion protein) were pooled and proteinconcentration was determined by BCA (Pierce) using bovine serum albuminas the standard.

Example 3 Expression of Pvs28 in Yeast

For expression in yeast, a Pvs28 DNA fragment was obtained by PCRamplification, as described above. A polyhistidine tag sequence wasspliced into the polypeptide coding sequence.

The resultant nucleic acid construct encoding a Pvs28 fusion protein(SEQ ID NO: 17) was ligated into the NheI and ApaI restriction sites ofthe yeast shuttle vector, YepRPEU-3 (as schematically represented inFIG. 6). Recombinant clones were electroporated into the host S.cerevisiae strain, VK1, and clones harboring the recombinant plasmidwere screened for their ability to secrete a His6 (SEQ ID NO:24) taggedprotein. High-producing colonies were amplified in selective growthmedia and used to establish cell banks for yeast expressed recombinantPvs28.

Example 4 Expression of Deglycosylated Pvs28 in Yeast

For expression in yeast, a Pvs28 DNA fragment was generated, asdescribed above. The nucleic acid was modified to encode a glutamine,rather than an asparagine, at amino acid residue number 130 (see FIG. 6,“Pvs28Q130”). A polyhistidine tag sequence was spliced into thepolypeptide coding sequence.

The resultant nucleic acid construct encoding this modified (partiallydeglycosylated) Pvs28 fusion protein (“Pvs28Q130”; SEQ ID NO: 18) wasligated into the NheI and Apal restriction sites of the yeast shuttlevector, YepRPEU-3 (as schematically represented in FIG. 6). Recombinantclones were electroporated into the host S. cerevisiae strain, VK1, andclones harboring the recombinant plasmid were screened for their abilityto secrete a His6 (SEQ ID NO: 15) tagged protein. High-producingcolonies were amplified in selective growth media and used to establishcell banks for yeast-expressed recombinant Pvs28.

A further variation of Pvs28 was generated (using similar techniques),as schematically represented in FIG. 6, see Pvs28NCR, and the amino acidsequence as represented by SEQ ID NO:19.

Example 5 Generation of High Titers of Antibodies Using RecombinantPvs25, Pvs28 and Deglycosylated Pvs28 Produced in a Yeast ExpressionSystem

This example demonstrates that the recombinant Pvs25 and Pvs28 of theinvention, generated in the yeast expression systems, as describedabove, can be used to used generate high titers of antigen specificantibodies in a mammal.

Recombinant Pvs25 and Pvs28 polypeptides were generated in the yeastexpression system as described above (see Examples 2 and 3, above).Immunogenic compositions comprising Pvs25 or Pvs28 and the adjuvant alumwere produced by standard methodologies. Briefly, 50 micrograms (ug) ofprotein was absorbed by 800 ug of alum in 500 microliters (ul) ofphosphate buffered saline (PBS) at pH 7.2. Purified recombinant proteinswere adsorbed to alum (Superfos Biosector a/s) for 30 min at roomtemperature with continuous rocking. The suspensions were then stored at4° C. until used to vaccinate mice by the intraperitoneal route.

Mice of various inbred strains, listed in FIG. 7 (which alsoschematically summarizes and illustrates the immunization protocol) wereused. Each was prebled before injection antigen (or alum control). Theanimals were then immunized intraperitoneally (IP) with either therecombinant Pvs25-containing or Pvs28-containing immunogeniccompositions; or alum alone. The mice were boosted with similarcompositions and dosages at day 21 and day 42. All mice were bled at day56. Harvested blood was prepared by standard protocols, and the testbleeds and pre-bleed controls were analyzed for anti-Pfs25 and Pvs28antibody titers using various standard immunological techniques, asdescribed above.

The antibody titer data clearly demonstrated high titers of anti-Pfs25antiserum were generated in all five strains of mice; the alum only testbleeds showed no anti-Pvs25 reactivity above background. Data alsoclearly demonstrated that high titers of anti-Pfs28 antiserum weregenerated in all four or the five strains of mice (only the C57BL/6strain did not respond to the Pvs28-containing immunogenic composition);the alum only test bleeds showed no anti-Pvs25 reactivity abovebackground.

Example 6 Anti-Pvs Antiserum have P. vivax Transmission BlockingActivity

Transmission-blocking activity was assayed as described previouslyQuakyi (1987) J. Immunol. 139: 4213-4217. Briefly, test sera were mixedwith mature in vitro-cultured P. vivax gametocytes and fed to mosquitoesthrough an artificial membrane stretched across the base of awater-jacketed glass cylinder. The parasites in the blood meal wereallowed to develop in the mosquito to the easily identifiable oocyststage by maintaining the mosquitoes in a secured insectary for 6-8 days.Infectivity was measured by dissecting the midgut, staining it withmercurochrome, and then counting the number of oocysts per mosquitomidgut of approximately 20 mosquitoes. The data was analyzed asdescribed in Kaslow et al Vaccine Res. 2:95-103.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A composition comprising an isolated Pvs25 polypeptide having atleast 95% amino acid sequence identity to SEQ ID NO:4, wherein the Pvs25polypeptide induces production of antibodies in a susceptible mammalagainst a 25 kD protein on the surface of Plasmodium vivax zygotes andookinetes, wherein the antibodies against the 25 kD protein block thetransmission of P. vivax from a mosquito.
 2. The composition of claim 1,wherein the Pvs25 polypeptide is encoded by a nucleic acid sequencehaving at least 95% sequence identity to SEQ ID NO:3.
 3. The compositionof claim 1, wherein the Pvs25 polypeptide has an amino acid sequence asshown in SEQ ID NO:4.
 4. The composition of claim 1, wherein thecomposition further comprises a Pvs28 polypeptide having at least 95%sequence identity to SEQ ID NO:2, wherein the Pvs28 polypeptide inducesproduction of antibodies in a susceptible mammal against a 28 kD proteinon the surface of Plasmodium vivax zygotes and ookinetes, wherein theantibodies against the 28 kD protein block the transmission of P.vivaxfrom a mosquito.
 5. The composition of claim 1, further comprisinga pharmaceutically acceptable carrier.
 6. The composition of claim 5,wherein the Pvs25 polypeptide comprises an amino acid having thesequence as set forth in SEQ ID NO:4.
 7. The composition of claim 5,wherein the composition further comprises a Pvs28 polypeptide having atleast 95% sequence identity to SEQ ID NO:2, wherein the Pvs28polypeptide induces production of antibodies in a susceptible mammalagainst a 28 kD protein on the surface of Plasmodium vivax zygotes andookinetes, wherein the antibodies against the 28 kD protein block thetransmission of P. vivaxfrom a mosquito.
 8. A method of inducingantibody production against Pvs25 on the surface of Plasmodium vivaxookinetes, the method comprising administering to a susceptible mammal apharmaceutical composition comprising a Pvs25 polypeptide having atleast 95% amino acid sequence identity to SEQ ID NO:4 in an amountsufficient to induce production of antibodies in the mammal against a 25kD protein on the surface of Plasmodium vivax zygotes and ookinetes,wherein the antibodies against the 25 kD protein block the transmissionof P. vivax from a mosquito.
 9. The method of claim 8, wherein the Pvs25polypeptide in the pharmaceutical composition is recombinantly produced.10. The method of claim 8, wherein the susceptible mammal is a human.11. The method of claim 8, wherein the Pvs25 polypeptide in thepharmaceutical composition is on the surface of a recombinant virus. 12.The method of claim 8, wherein the Pvs25 polypeptide is encoded by anucleic acid having at least 95% nucleic acid sequence identity to SEQID NO:3.
 13. The method of claim 12, wherein the Pvs25 polypeptidecomprises an amino acid having the sequence as set forth in SEQ ID NO:4.14. The method of claim 8, further comprising administering a Pvs28polypeptide having at least 95% amino acid sequence identity to SEQ IDNO:2, wherein the Pvs28 polypeptide induces production of antibodies ina susceptible mammal against a 28 kD protein on the surface ofPlasmodium vivax zygotes and ookinetes, wherein the antibodies againstthe 28 kD protein block the transmission of P. vivax from a mosquito.15. The method of claim 8, wherein the Pvs25 polypeptide is administeredintramuscularly, intradermally, subcutaneously, or intranasally.
 16. Themethod of claim 14, wherein the Pvs25 polypeptide and the Pvs28polypeptide are administered as a fusion protein having at least 95%amino acid sequence identity to SEQ ID NO:5.
 17. The composition ofclaim 4, wherein the Pvs25 polypeptide and the Pvs28 polypeptide are afusion protein having at least 95% amino acid sequence identity to SEQID NO:5.